Technical Field
[0001] The present invention relates to a fusion protein which has an antigen-binding function
and is expressed in an intracellular environment.
Background Art
[0002] Intracellularly functionable antibodies, i.e., intrabodies are capable of affecting
functions of cells of a higher organism by recognizing and binding to antigens (target
molecules) in the cells. These antigens can be important intracellular therapeutic
targets which can be inactivated upon binding to intrabodies. Further, in terms of
research technique, use of intrabodies has been attracting attention as means for
directly and specifically inhibiting functions of proteins by binding to antibodies
inside cells.
[0003] To produce an intrabody, it is typical to (i) first use a standard method to prepare
monoclonal-antibody-producing hybridomas capable of recognizing an antigen, (ii) then
construct, from cDNA of each monoclonal-antibody-producing hybridoma, an intracellular
expression vector containing DNA encoding a single chain Fv (scFv), and (iii) thus
using a complex of heavy chain (VH) and light chain (VL) as an intrabody.
[0004] An antibody ordinarily functions by circulating in an extracellular environment (e.g.,
blood) inside the body and recognizing an extracellular antigen. It is thus assumed
that an antibody acts in an extracellular environment. As such, in a case where an
antibody is expressed in cytoplasm, the antibody suffers folding and stability issues
leading to a lower expression level and a limited half-life of a domain of the antibody.
It thus cannot be expected that any antibody functions as an intrabody. Further, scFv
is constituted by a heavy chain and a light chain which are connected by a flexible
peptide linker, and thus has a tertiary structure not as stable as an original antibody.
Furthermore, even if scFv that functions in a test tube is identified, that scFv may
not necessarily able to exhibit an expected function when expressed in a cell, since
many aspects of an intracellular environment are not reflected in the environment
inside the test tube.
[0005] Thus, there have been conducted studies on (i) regularity among antibodies capable
of withstanding cytoplasmic conditions and (ii) a technique for expecting such antibodies,
in order to enable screening of an antibody that functions as an intrabody. These
are methods for selecting an antibody capable of functioning in cells. It is difficult,
however, to actually obtain an intracellularly stable antibody with use of these methods.
There has not been established a technology that utilizes an existing antibody or
a newly isolated antibody as an intrabody.
Citation list
[Patent Literature]
[0006] [Patent Literature 1]
International Publication No.
WO 03/014960
Summary of Invention
Technical Problem
[0007] It is an object of the present invention to provide a technology for using a given
antigen-binding peptide as an intrabody that stably functions in a cell.
Solution to Problem
[0008] The inventors of the present invention conducted diligent study in order to attain
the object. As a result, the inventors discovered that when an antigen-binding peptide
fused with a peptide tag is expressed in a cell, functional stability of the antigen-binding
peptide as an intrabody changes in accordance with changes caused by the peptide tag
in values of net charge and isoelectric point (pI) of the antigen-binding peptide.
In particular, by designing the peptide tag so that values of charge and pI of the
intrabody are sufficiently low with respect to a pH environment on a cytoplasmic surface
of an endosome rather than a pH environment of the cytoplasm, the inventors achieved
that the antigen-binding peptide stably functions as an intrabody in a cell by being
fused with that peptide tag.
[0009] Based on the above knowledge, the inventors of the present invention further conducted
study and consequently completed the present invention.
[0010] That is, the present invention is as follows:
- 1) A fusion protein, containing:
an intracellular-stabilizer peptide; and an antigen-binding peptide,
the intracellular-stabilizer peptide consisting of 10 to 39 amino acids, at least
45% of the 10 to 39 amino acids being acidic amino acids,
the antigen-binding peptide containing at least one of heavy chain CDR1, heavy chain
CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3.
- 2) The fusion protein as set forth in 1), wherein:
28% or less of the 10 to 39 amino acids of the intracellular-stabilizer peptide are
basic amino acids; or
the intracellular-stabilizer peptide contains no basic amino acid.
- 3) The fusion protein as set forth in 2), wherein the intracellular-stabilizer peptide
contains no basic amino acid.
- 4) The fusion protein as set forth in 2), wherein the intracellular-stabilizer peptide
contains at least one histidine as a basic amino acid.
- 5) The fusion protein as set forth in any one 1) through 4), wherein:
the intracellular-stabilizer peptide consists of 14 to 25 amino acids, at least 50%
of the 14 to 25 amino acids being acidic amino acids; and
the intracellular-stabilizer peptide containing neither a portion consisting of 8
or more consecutive acidic amino acids nor a portion consisting of 4 or more consecutive
amino acids that are not acidic amino acids.
- 6) The fusion protein as set forth in 5), wherein the intracellular-stabilizer peptide
contains neither a portion consisting of 3 or more consecutive acidic amino acids
nor a portion consisting of 3 or more consecutive amino acids that are not acidic
amino acids.
- 7) The fusion protein as set forth in 1), wherein the intracellular-stabilizer peptide
contains
the following amino acid sequence (1) or (2):
or -Xn-XA-Xn-XA-Xn-XA-XA-Xn-XA-Xn-Xn-XA-XA-Xn-Xn-XA- ...(2),
where XA is an acidic amino acid and Xn is an amino acid that is not an acidic amino acid.
- 8) The fusion protein as set forth in 7), wherein XA is aspartic acid or glutamic acid, and Xn is an amino acid selected from the group consisting of asparagine, glutamine, proline,
tyrosine, and valine.
- 9) A polynucleotide encoding a fusion protein recited in any one of 1) through 8)
above.
- 10) An expression vector containing a polynucleotide recited in 9) above.
- 11) A method for producing a cell capable of expressing an antigen-binding peptide
in the cell, the method including the step of: introducing a polynucleotide recited
in 9) or an expression vector recited in 10) into a cell.
- 12) A cell capable of expressing a fusion protein recited in any one of 1) through
8) above.
Brief Description of Drawings
[0011]
Fig. 1 is a view illustrating results of evaluation of the expression and binding
capacity of scFv-A36.
Fig. 2 is a view illustrating information of the amino acid sequence of CDR in VH region of scFv-A36.
Fig. 3 is a view illustrating results of evaluation of (i) the expression of scFv-GFPA36
and scFv-GFPM4 in cultured neurons and (ii) the activity of scFv-GFPA36 and scFv-GFPM4
to inhibit the function of a target molecule.
Fig. 4 is a view illustrating confirmation of aggregation of scFv-GFPA36 in neurons
of mouse brain.
Fig. 5 is a view illustrating the pI and net charge of scFv-A36 and scFv-M4 and results
of evaluation of the intracellular aggregation property of scFv-A36 and scFv-M4.
Fig. 6 is a view illustrating results of examination of the generality of the effects
of s3Flag- and HA-peptide tagging on the increase in the net negative charge of intrabodies
at the lower pH using other 94 scFv protein sequences obtained by NCBI blast search
by using of scFv-A36 protein sequence as a query sequence.
Fig. 7 is a view illustrating results of examination of the biochemical property of
s3Flag-scFvA-36-HA construct.
Fig. 8 is a view illustrating results of examination of intracellular expression of
s3Flag-scFv-A36-HA and s3Flag-scFv-M4-HA in neurons of mouse brain.
Fig. 9 is a view illustrating results of examination of the expression patterns of
s3Flag-scFv-A36-HA and s3Flag-scFv-M4-HA in substantia nigra region.
Fig. 10 is a view illustrating results of observation of long-term (6 months) expression
of both s3Flag-scFv-A36-HA and s3Flag-scFv-M4-HA in dopamine neurons.
Fig. 11 is a view illustrating results of examination of the functional activity of
s3Flag-scFv-A36-HA against Syt I in dopamine neurons in vivo.
Fig. 12 is a view illustrating results of motor behavior test and immunohistological
staining of s3Flag-scFv-A36-HA-expressing mice and s3Flag-scFv-M4-HA-expressing mice.
Fig. 13 is a view illustrating results of examination of the expression and antitumor
activity of STAND-Y13-259.
Fig. 14 is a view illustrating results of examination of the expression and antitumor
activity of DE2.0-Y13-259-HA.
Fig. 15 is a view illustrating results of examination of a change in the net negative
charge of scFv-6E, caused by fusion of scFv-6E and a tag.
Fig. 16 is a view illustrating results of observation of intracellular expression
of STAND-6E-LYS and DE5.0-6E.
Fig. 17 a view illustrating results of α-synuclein aggregation assay.
Fig. 18 a view illustrating results of evaluation of α-synuclein aggregation.
Description of Embodiments
<0. Definition>
(Peptide)
[0012] In the present specification, the term "peptide" is interchangeable with "polypeptide"
or "protein". A "peptide" includes a structure of amino acids linked by a peptide
bond. A "peptide" may further include, for example, a structure of a sugar chain,
an isoprenoid group, or the like. The term "peptide", unless otherwise specified,
includes in its scope a peptide that contains an already known analog of a naturally-occurring
amino acid having a function as well as the naturally-occurring amino acid.
(Acidic amino acid)
[0013] In the present specification, the term "acidic amino acid" refers to an amino acid
having an isoelectric point of 3.99 or less. This amino acid may be a naturally-occurring
amino acid or an analog of a naturally-occurring amino acid. Examples of a naturally-occurring
acidic amino acid include aspartic acid and glutamic acid.
(Basic amino acid)
[0014] In the present specification, the term "basic amino acid" refers to an amino acid
having an isoelectric point of 7.40 or more. This amino acid may be a naturally-occurring
amino acid or an analog of a naturally-occurring amino acid. Examples of a naturally-occurring
basic amino acid include histidine, lysine, and arginine.
(Amino acid that is neither acidic amino acid nor basic amino acid)
[0015] In the present specification, an amino acid that fits neither the above definition
of an acidic amino acid nor the above definition of a basic amino acid in terms of
pH of the amino acid itself is referred to generally as "an amino acid that is neither
an acidic amino acid nor a basic amino acid" or "a substantially neutral amino acid".
That is, such an amino acid has an isoelectric point of more than 3.99 and less than
7.40. The amino acid may be a naturally-occurring amino acid or an analog of a naturally-occurring
amino acid.
(A and/or B)
[0016] In the present specification, the expression "A and/or B" is a concept covering both
"A and B" and "A or B", and is interchangeable with "at least one of A and B".
<1. Fusion protein>
[0017] A fusion protein of the present invention is a fusion protein containing:
an intracellular-stabilizer peptide; and
an antigen-binding peptide,
the intracellular-stabilizer peptide consisting of 10 to 39 amino acids, at least
45% of the 10 to 39 amino acids being acidic amino acids,
the antigen-binding peptide containing at least one of heavy chain CDR1, heavy chain
CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3.
[0018] The fusion protein of the present invention serves as an intrabody by being expressed
in a given cell.
[Intracellular-stabilizer peptide]
(Peptide length and containing acidic amino acids)
[0019] The intracellular-stabilizer peptide can be constituted by any number of amino acids.
The intracellular-stabilizer peptide is preferably a peptide consisting of 50 or less
amino acids, more preferably a peptide consisting of 10 to 39 amino acids. Note that
at least 45% of all amino acids constituting the intracellular-stabilizer peptide
are acidic amino acids.
[0020] A percentage of the acidic amino acids among all amino acids constituting the intracellular-stabilizer
peptide is not particularly limited, but is preferably 50% or more. The percentage
of the acidic amino acids among all amino acids constituting the intracellular-stabilizer
peptide may be, for example, 53% or more or 55% or more. The percentage may be preferably
60% or more, and may be preferably 65% or more or 70% or more. In general, an isoelectric
point of a polypeptide containing acidic amino acids decreases as a percentage of
the acidic amino acids increases.
[0021] The number of the acidic amino acids constituting the intracellular-stabilizer peptide
is not particularly limited, provided that any of the above ranges of percentage of
the acidic amino acids among all amino acids constituting the intracellular-stabilizer
peptide is met. For example, the number of the acidic amino acids constituting the
intracellular-stabilizer peptide is 6 or more or 7 or more, preferably 8 or more or
9 or more, more preferably 10 or more, even more preferably 11 or more, and particularly
preferably 12 or more.
[0022] The acidic amino acids constituting the intracellular-stabilizer peptide are each
preferably selected from the group consisting of aspartic acid and glutamic acid.
[0023] From the viewpoint of increasing a degree of contribution of the intracellular-stabilizer
peptide in the fusion protein, the number of the amino acids constituting the intracellular-stabilizer
peptide is preferably 14 or more, more preferably 15 or more, even more preferably
18 or more, and particularly preferably 20 or more. Further, from the viewpoint of
reducing a size of the fusion protein, the number of the amino acids constituting
the intracellular-stabilizer peptide is preferably 35 or less, more preferably 30
or less, and even more preferably 25 or less. Note that in a case where a length (the
number of the amino acids constituting the intracellular-stabilizer peptide) of the
intracellular-stabilizer peptide is 9 amino acids or less, the intracellular-stabilizer
peptide may be inadequate from the viewpoint of contribution of the intracellular-stabilizer
peptide in the fusion protein. Further, the intracellular-stabilizer peptide having
a length of 9 amino acids or less may be inadequate also from the viewpoint of antigen-specificity
which the intracellular-stabilizer peptide is required to have when necessary, for
example, when an antibody capable of recognizing the intracellular-stabilizer peptide
is to be produced. Further, in a case where the intracellular-stabilizer peptide has
a length of 40 amino acids or more, the fusion protein has an unnecessarily large
size, and can accordingly cause steric hindrance in antigen recognition by the antigen-binding
peptide (described later). Note, however, that the intracellular-stabilizer peptide
having a length of 40 amino acids or more can be used in a case where a peptide serving
as a linker is provided between and linked to the intracellular-stabilizer peptide
and the antigen-binding peptide so that the intracellular-stabilizer peptide does
not interfere with the antigen recognition. In that case, a plurality of intracellular-stabilizer
peptides (which may be a plurality of intracellular-stabilizer peptides of the same
kind or may be a combination of different kinds of intracellular-stabilizer peptides)
may be used by being linked to one another via a spacer between each adjacent ones
of the intracellular-stabilizer peptides.
(Case in which basic amino acid is contained)
[0024] A percentage of basic amino acids among all amino acids constituting the intracellular-stabilizer
peptide is not particularly limited, and is preferably 28% or less, more preferably
27% or less or 26.5% or less, even more preferably 25% or less, 20% or less, 15% or
less, or 10% or less, and particularly preferably 9% or less, 8% or less, 7% or less,
6% or less, 5% or less, 4% or less, or 3% or less. In a particularly preferable embodiment,
the intracellular-stabilizer peptide contains no basic amino acid.
[0025] A preferable range of the number of basic amino acids constituting the intracellular-stabilizer
peptide is not particularly limited, provided that any of the above ranges of percentage
of acidic amino acids among all amino acids constituting the intracellular-stabilizer
peptide is met. For example, the number of basic amino acids constituting the intracellular-stabilizer
peptide is 6 or less, preferably 5 or less or 4 or less, more preferably 3 or less,
even more preferably 2 or less, and particularly preferably 1 or less.
[0026] In a case where the intracellular-stabilizer peptide contains a basic amino acid,
it is preferable that the basic amino acid be selected from the group consisting of
lysine and histidine, and it is more preferable that the intracellular-stabilizer
peptide contain at least one histidine as the basic amino acid. Histidine has a characteristic
of having the lowest isoelectric point among naturally-occurring basic amino acids.
In a case where the intracellular-stabilizer peptide contains a plurality of basic
amino acids, a percentage of histidine among the basic amino acids is preferably 30%
or more, more preferably 50% or more, even more preferably 60% or more, and particularly
preferably 100%. The number of histidine residues contained in the intracellular-stabilizer
peptide is, for example, 6, 5, 4, 3, 2, or 1.
(Amino acid that is neither acidic amino acid nor basic amino acid)
[0027] The intracellular-stabilizer peptide may contain an amino acid (generally referred
to as a "substantially neutral amino acid) that is neither an acidic amino acid nor
a basic amino acid, which have been described above.
[0028] A percentage of substantially neutral amino acids among all amino acids constituting
the intracellular-stabilizer peptide is 55% or less, preferably 50% or less, and more
preferably 45% or less. In a case where the intracellular-stabilizer peptide contains
a substantially neutral amino acid, a lower limit of a percentage of the substantially
neutral amino acid among all amino acids constituting the intracellular-stabilizer
peptide is not particularly limited, and is, in an aspect, 15% or more, 20% or more,
or 25% or more.
[0029] When characteristics of the intracellular-stabilizer peptide as an antigen are taken
into consideration, a percentage of hydrophobic amino acids (i.e., nonpolar, substantially
neutral amino acids) among all amino acids constituting the intracellular-stabilizer
peptide is preferably 25% or less, more preferably 20% or less, and even more preferably
15% or less. However, when a hydrophilic amino acid-hydrophobic amino acid balance
in the intracellular-stabilizer peptide is taken into consideration, it may be preferable
that one or more hydrophobic amino acids are contained in the intracellular-stabilizer
peptide, on the premise that the above range of percentage of hydrophobic amino acids
is met. In a case where the intracellular-stabilizer peptide contains hydrophobic
amino acids, the number of the hydrophobic amino acids is, for example, 1, 2, 3, or
4. It may be preferable that the number of consecutive hydrophobic amino acids be
3 or less, and it may be more preferable that the number of consecutive hydrophobic
amino acids be 2 or less.
[0030] In a case where the intracellular-stabilizer peptide contains a naturally-occurring
substantially neutral amino acid, the substantially neutral amino acid is preferably
selected from the group consisting of asparagine, phenylalanine (a hydrophobic amino
acid), glutamine, tyrosine, serine, methionine (a hydrophobic amino acid), tryptophan
(a hydrophobic amino acid), valine (a hydrophobic amino acid), glycine (a hydrophobic
amino acid), leucine (a hydrophobic amino acid), isoleucine (a hydrophobic amino acid),
and proline (a hydrophobic amino acid), more preferably selected from the group consisting
of asparagine, glutamine, tyrosine, methionine, valine, glycine, leucine, isoleucine,
and proline, and even more preferably selected from the group consisting of asparagine,
glutamine, tyrosine, valine, and proline. In an aspect, the intracellular-stabilizer
peptide contains at least one (preferably 1 or 2) proline, at least one (preferably
8 or less, for example, 7, 6, 5, 4, 3, or 2) amino acid selected from asparagine,
glutamine, and tyrosine, and at least one (preferably 1 or 2) hydrophobic amino acid
that is not proline.
[0031] Note that preferable types and arrangement of substantially neutral amino acids may
be determined, for example, by taking account of: stability (resistance to oxidization,
hydrolysis, and the like) of the intracellular-stabilizer peptide formed; whether
or not an intramolecular S-S bond is formed; whether or not an intramolecular hydrogen
bond is formed; whether or not an undesired motif is formed; and the like.
(Isoelectric point)
[0032] An isoelectric point of the intracellular-stabilizer peptide can be calculated from
types of amino acids constituting the intracellular-stabilizer peptide. Calculation
of the isoelectric point of the intracellular-stabilizer peptide can be carried out,
for example, in accordance with descriptions of Protein Caluculator (http://protcalc.sourceforge.net)
or the like.
(More specific examples of intracellular-stabilizer peptide)
[0033] The following description will discuss more specific examples of the intracellular-stabilizer
peptide. Note that in the description below, X
A refers to an acidic amino acid, and X
n refers to an amino acid that is not an acidic amino acid, i.e., X
n refers to a basic amino acid and a substantially neutral amino acid, which have been
described above.
[0034] Note that all of the descriptions in the section [Intracellular-stabilizer peptide]
apply to the specific examples below of the intracellular-stabilizer peptide. For
example, all of the above descriptions of an acidic amino acid apply to X
A, and all of the above descriptions of a basic amino acid and a substantially neutral
amino acid apply to X
n. Also with regard to a length etc. of the intracellular-stabilizer peptide, all of
the above descriptions apply. Specifically, for example, the examples below of the
intracellular-stabilizer peptide may each be an intracellular-stabilizer peptide in
which 28% or less of the amino acids are basic amino acids or be an intracellular-stabilizer
peptide which contains no basic amino acid (i.e., all X
n are substantially neutral amino acids). Further, the intracellular-stabilizer peptide
may be an intracellular-stabilizer peptide that contains at least one histidine as
a basic amino acid.
(1) Aspect 1
[0035] An intracellular-stabilizer peptide that satisfies all of the conditions listed below.
- The intracellular-stabilizer peptide consists of 10 to 39 amino acids and preferably
consists of 14 to 25 amino acids.
- Of the amino acids constituting the intracellular-stabilizer peptide, at least 45%
are acidic amino acids, and preferably at least 50% are acidic amino acids.
- The intracellular-stabilizer peptide contains neither a portion consisting of 8 or
more consecutive XA nor a portion consisting of 4 or more consecutive Xn. That is, in the intracellular-stabilizer peptide, the number of consecutive XA is 7 or less and the number of consecutive Xn is 3 or less.
(2) Aspect 2
[0036] An intracellular-stabilizer peptide that satisfies the conditions of Aspect 1 above
and satisfies the condition listed below.
- The intracellular-stabilizer peptide contains neither a portion consisting of 6 or
more consecutive XA nor a portion consisting of 4 or more consecutive Xn. That is, in the intracellular-stabilizer peptide, the number of consecutive XA is 5 or less and the number of consecutive Xn is 3 or less.
(3) Aspect 3
[0037] An intracellular-stabilizer peptide that satisfies the conditions of Aspect 1 above
and satisfies the condition listed below. - The intracellular-stabilizer peptide contains
neither a portion consisting of 6 or more consecutive X
A nor a portion consisting of 3 or more consecutive X
n. That is, in the intracellular-stabilizer peptide, the number of consecutive X
A is 5 or less and the number of consecutive X
n is 2 or less.
(4) Aspect 4
[0038] An intracellular-stabilizer peptide that satisfies the conditions of Aspect 1 above
and satisfies the condition listed below.
- The intracellular-stabilizer peptide contains neither a portion consisting of 3 or
more consecutive XA nor a portion consisting of 3 or more consecutive Xn. That is, in the intracellular-stabilizer peptide, the number of consecutive XA is 2 or less and the number of consecutive Xn is 2 or less.
(5) Aspect 5
[0039] An intracellular-stabilizer peptide that satisfies all of the conditions listed below.
Note that Aspect 5 may or may not fall under any of Aspects 1 to 4.
- The intracellular-stabilizer peptide consists of 10 to 39 amino acids and preferably
consists of 14 to 25 amino acids.
- Of the amino acids constituting the intracellular-stabilizer peptide, at least 45%
are acidic amino acids, and preferably at least 50% are acidic amino acids.
- The intracellular-stabilizer peptide contains the following amino acid sequence:
(6) Aspect 6
[0040] An intracellular-stabilizer peptide that satisfies the conditions of Aspect 5 above,
has 3 to 7 more X
A or X
n on a left side of the amino acid sequence (1) shown in Aspect 5, and has 0 to 6 more
X
A or X
n on a right side of the amino acid sequence (1) shown in Aspect 5.
(7) Aspect 7
[0041] An intracellular-stabilizer peptide that satisfies all of the conditions listed below.
Note that Aspect 7 may or may not fall under any of Aspects 1 to 4.
- The intracellular-stabilizer peptide consists of 10 to 39 amino acids and preferably
consists of 14 to 25 amino acids.
- Of the amino acids constituting the intracellular-stabilizer peptide, at least 45%
are acidic amino acids, and preferably at least 50% are acidic amino acids.
- The intracellular-stabilizer peptide contains the following amino acid sequence:
-Xn-XA-Xn-XA-Xn-XA-XA-Xn-XA-Xn-Xn-XA-XA-Xn-Xn-XA- ...(2).
(8) Aspect 8
[0042] An intracellular-stabilizer peptide that satisfies the conditions of Aspect 7 above,
has 0 to 9 more X
A or X
n on a left side of the amino acid sequence (2) shown in Aspect 7, and has 0 to 9 more
X
A or X
n on a right side of the amino acid sequence (2) shown in Aspect 7. Note, however,
that a total of the numbers of the amino acids on the respective left and right sides
of the amino acid sequence (2) may be preferably 10 or less, more preferably 9 or
less, and even more preferably 6 or less or 5 or less.
(9) Aspect 9
[0043] An intracellular-stabilizer peptide that falls under any one of Aspects 1 to 8 above,
wherein X
A is aspartic acid or glutamic acid, and X
n is an amino acid selected from the group consisting of asparagine, glutamine, proline,
tyrosine, and valine.
(10) Aspect 10
[0044] An intracellular-stabilizer peptide that falls under any one of Aspects 5 to 6 above,
wherein: X
A is aspartic acid or glutamic acid; and among X
n, (i) one or more of 2th to 6th (preferably 2th to 4th, more preferably 2nd to 3rd)
X
n from the left side of the amino acid sequence (1) in the amino acid sequence (1)
are each proline and (ii) each X
n that is not proline is an amino acid selected from the group consisting of asparagine,
glutamine, tyrosine, and valine. As an example, one of the 2nd to 3rd X
n from the left side of the amino acid sequence (1) in the amino acid sequence (1)
is proline, and the other X
n contained in the amino acid sequence (1) are each an amino acid selected from the
group consisting of asparagine, glutamine, valine, and tyrosine. As another example,
one of the 2nd to 3rd X
n from the left side of the amino acid sequence (1) in the amino acid sequence (1)
is proline, and the other X
n contained in the amino acid (1) are each an amino acid selected from the group consisting
of asparagine, glutamine, and tyrosine.
(11) Aspect 11
[0045] An intracellular-stabilizer peptide that falls under any one of Aspects 7 to 8 above,
wherein: X
A is aspartic acid or glutamic acid; and among X
n, (i) one or more of 3rd to 6th (preferably 3rd to 5th, more preferably 4th to 5th)
X
n from the left side of the amino acid sequence (2) in the amino acid sequence (2)
are each proline and (ii) each X
n that is not proline is an amino acid selected from the group consisting of asparagine,
glutamine, tyrosine, and valine. As an example, one of the 4th to 5th X
n from the left side of the amino acid sequence (2) in the amino acid sequence (2)
is proline, the other of the 4th to 5th X
n is valine, and the other X
n contained in the amino acid sequence (2) are each an amino acid selected from the
group consisting of asparagine, glutamine, and tyrosine.
(12) Aspect 12
[0046] An intracellular-stabilizer peptide consists of any one of the following amino acid
sequences.
[0047]
MDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 1);
EEDQDDEDDEDQDD (SEQ ID NO: 2);
NDEYEDPDEQDDEND (SEQ ID NO: 3);
QDEVDEPEDEEDNDD (SEQ ID NO: 4);
QDEVDEPEDEDENDD (SEQ ID NO: 5);
QDEVDEPEDEDENQD (SEQ ID NO: 6);
QDNVDEPEDNDENQD (SEQ ID NO: 7);
QDNYDEPEDNDENQD (SEQ ID NO: 8);
EDNYDEPEDNDENQD (SEQ ID NO: 9);
DNNYDEQDENEQPED (SEQ ID NO: 10);
QENDYDEPEVNDENQD (SEQ ID NO: 11);
DEQENDYDEPEVNDENQD (SEQ ID NO: 12); and
DEQENDYDEPEVNDENQDYDE (SEQ ID NO: 13).
(13) Aspect 13
[0048] An intracellular-stabilizer peptide that satisfies all of the conditions listed below.
- The intracellular-stabilizer peptide consists of 10 to 39 amino acids and preferably
consists of 14 to 25 amino acids.
- All amino acids constituting the intracellular-stabilizer peptide are acidic amino
acids.
(Antigen-binding peptide)
(Structure)
[0049] A typical antibody structural unit is known to include a tetramer. Each tetramer
is constituted by two identical pairs of polypeptide chains, each pair having one
light chain (e.g., approximately 25 kDa) and one heavy chain (e.g., approximately
50 kDa to 70 kDa). The amino-terminal portion of each chain has a variable region
of approximately 100 or more amino acids, and the variable region is primarily responsible
for antigen (target molecule) recognition. The carboxy-terminal portion of each chain
defines a constant region primarily responsible for effector function. Light chains
are classified as either kappa or lambda. Heavy chains are classified as gamma, mu,
alpha, delta, or epsilon, which in turn define the isotypes IgG, IgM, IgA, IgD and
IgE, respectively, of the antibody. Within the light chain and the heavy chain, the
variable region and the variable region are linked to each other by a J region of
approximately 12 or more amino acids, and the heavy chain also includes a D region
of approximately 10 more amino acids. The variable regions of each light/heavy chain
pair form an antibody binding site. These chains all represent the same general structure
of relatively conserved framework regions (FRs) linked to one another via three hypervariable
regions (also referred to as complementarity determining regions, or CDRs). CDRs derived
from the two chains of each pair are arranged by framework regions, so that binding
to a specific epitope is made possible.
[0050] The term "antigen-binding peptide" refers to a peptide which (i), in a given monoclonal
antibody or in a peptide capable of specifically binding to an antigen (a target molecule)
similarly as a monoclonal antibody, contributes to binding to the antigen and (ii)
has all or part of a region of the antibody which region has an antigen binding capacity.
In the present invention, the "antigen-binding peptide", for example, contains at
least one of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1,
light chain CDR2, and light chain CDR3 of a given monoclonal antibody. It is preferable
that the antigen-binding peptide contains at least 2, at least 3, at least 4, at least
5, or all of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1,
light chain CDR2, and light chain CDR3.
[0051] A monoclonal antibody from which the antigen-binding peptide of the present invention
is produced may be a natural antibody or an antibody which is produced by genetic
recombinant technology. The natural antibody is not limited to any particular one,
and can be derived from various species of organism such as a human, a mouse, a rat,
a monkey, a goat, a rabbit, a camel, a llama, a cow, and a chicken. The antibody produced
by the genetic recombinant technology is not limited to any particular one, and can
be, for example, (i) a synthesized antibody produced from a natural antibody, (ii)
a recombinant antibody, or (iii) a mutated antibody. Examples of the antibody also
include antibodies obtained by subjecting antibodies already produced by genetic recombinant
technology to modification that is similar to the aforementioned genetic modification
of natural antibodies. The antigen-binding peptide of the present invention is a peptide
which includes the full length or part of F(ab')
2, F(ab'), Fab', Fab, Fv (variable fragment of antibody), scFv, dsFv (disulphide stabilized
Fv), dAb (single domain antibody), a diabody, a minibody, or VHH derived from any
of the above antibodies. Examples of the "peptide capable of specifically binding
to an antigen similarly as a monoclonal antibody" include: a peptide aptamer having
high binding specificity for an antigen; a binding domain of a protein (e.g., fibronectin)
derived from a living organism having binding specificity for a target molecule; and
the like.
[Additional functional peptide]
[0052] The fusion protein of the present invention may further contain one or more functional
peptides other than the intracellular-stabilizer peptide and the antigen-binding peptide.
Examples of such a functional peptide include a purification tag peptide, a detection
peptide, a degradation promoting peptide (e.g., an HSC70 binding peptide, an XIAP
RING domain peptide), a Neh2 domain peptide (a peptide derived from Nrf2), a stress-responsive
degradation peptide such as an oxygen-dependent degradation domain (a peptide derived
from Hif1α), a drug-binding peptide, and the like.
[0053] The purify tag peptide is not limited to any particular one, and can be a purification
tag peptide containing acidic amino acids in a proportion of 15% or more, such as
an HA tag sequence (YPYDVPDYA (SEQ ID NO: 14)) and a PA tag sequence (GVAMPGAEDDVV
(SEQ ID NO: 15)). In a case where the fusion protein contains the tag sequence containing
acidic amino acids in a proportion of 15% or more, a further increase in negative
charge of the fusion protein is achieved also at low pH. It is more preferable that
the purification tag peptide contain acidic amino acids in a proportion of 20% or
more.
[0054] The detection peptide is not limited to any particular one, and can be, for example,
a fluorescent protein, an enzyme which emits light or undergoes a change in color
through a reaction, or the like. The fluorescent protein can be, but is not limited
to, BFP, EBFP, CFP, ECFP, Cypet, AmCyan1, GFP, EGFP, YFP, Venus, mKO, mOrange, RFP,
DsRed, tdTomato, mcherry, mStrawberry, Azalea, mPlum, mAG, Kaede, Dronpa, Keima, KikG,
KikGR, UnaG or the like. The enzyme which emits light or undergoes a change in color
through a reaction can be, but is not limited to, peroxidase, alkaline phosphatase,
β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase,
malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase,
acetylcholinesterase, or the like.
[Structure of fusion protein]
[0055] The term "fusion protein" refers to a protein which has at least two different types
of peptides that are linked to each other by a covalent bond, directly or via a linker.
The linker is not limited to any particular one. In order to impart flexibility, the
linker preferably mainly contains, for example, an amino acid having a low-molecular
side chain, such as glycine, alanine, and serine. It is preferable that 80%, 90%,
or more of the linker sequence contain a glycine residue, an alanine residue, or a
serine residue, and it is particularly preferable that 80%, 90%, or more of the linker
sequence contain a glycine residue or a serine residue.
[0056] The intracellular-stabilizer peptide and the antigen-binding peptide contained in
the fusion protein may be linked to each other in any order. The intracellular-stabilizer
peptide and the antigen-binding peptide may be arranged in this order from the N-terminal
side of the fusion protein, or the antigen-binding peptide and the intracellular-stabilizer
peptide may be arranged in this order from the N-terminal side of the fusion protein.
Further, the intracellular-stabilizer peptide and the antigen-binding peptide may
be arranged such that a direction of the N-terminal to the C-terminal of the fusion
protein is opposite to a direction of the N-terminal to the C-terminal of the intracellular-stabilizer
peptide and/or the antigen-binding peptide themselves. Further, a plurality of intracellular-stabilizer
peptides (which may be a plurality of intracellular-stabilizer peptides of the same
type or may be a combination of intracellular-stabilizer peptides of different types)
may be arranged such that each of the plurality of intracellular-stabilizer peptides
is respectively provided on both ends of the antigen-binding peptide.
[0057] The additional functional peptide may also be provided in any arrangement. In a case
where the functional peptide is a purification tag peptide containing acidic amino
acids in a proportion of 15% or more, the purification tag peptide is preferably arranged
such that the antigen-binding peptide is interposed between the purification tag peptide
and the intracellular-stabilizer peptide ("the intracellular-stabilizer peptide -
the antigen-binding peptide - the purification tag peptide" or "the purification tag
peptide - the antigen-binding peptide - the intracellular-stabilizer peptide). This
arrangement allows achieving a positional balance of overall charge of the fusion
protein. Accordingly, it becomes possible to maintain the fusion protein more stable
in a cell. The detection peptide may also be provided in any arrangement. From the
viewpoint of detectability, it is preferable that the detection peptide be located
at the very N-terminal or the very C-terminal of the fusion protein.
[0058] Further, identical or different intracellular-stabilizer peptides may be arranged
such that each of the intracellular-stabilizer peptides is respectively linked to
both ends (i.e., the N-terminal and the C-terminal) of the antigen-binding peptide.
In this case, the fusion protein has an increased negative charge also at lower pH.
This enables more successfully preventing aggregation in cells.
[0059] The fusion protein of the present invention, due to containing the intracellular-stabilizer
peptide, has a low isoelectric point as compared with a case in which the antigen-binding
peptide is present alone. This allows the fusion protein to undergo less aggregation
in a cell as compared with a case in which the antigen-binding peptide is expressed
alone in a cell. Thus, the fusion protein can stably function as an intrabody.
In an example, the fusion protein of the present invention preferably has a net negative
charge at pH 7.4, more preferably has a net negative charge at pH 6.6, more preferably
has a net negative charge at pH 6.0, and even more preferably has a net negative charge
at pH 5.0.
[0060] The number of amino acids constituting the "fusion protein" is not particularly limited,
and is, for example, 1000 or less, preferably 600 or less, more preferably 550 or
less. The number of amino acids constituting the "fusion protein" is not particularly
limited, and is, for example, 100 or more, 250 or more, or 300 or more.
<2. Polynucleotide>
[0061] A polynucleotide of the present invention encodes the fusion protein of the present
invention. The term "polynucleotide" may refer to any of a DNA molecule, an RNA molecule,
and a hybrid molecule of DNA and RNA. The term "polynucleotide" may refer to a double-stranded
polynucleotide or a single-stranded polynucleotide.
[0062] The polynucleotide of the present invention can be prepared by a well-known genetic
engineering technique, chemical synthesis method, or the like. A specific base sequence
of the polynucleotide of the present invention can be easily designed by a person
skilled in the art from an amino acid sequence of an intended fusion protein and with
reference to, for example, the codon table.
[0063] Further, the polynucleotide of the present invention may contain: a regulatory sequence
such as a promoter (SV40 promoter, MMTV-LTR promoter, EF1α promoter, CMV promoter,
or the like), an enhancer, a ribosome binding site, a splice signal, and a terminator;
a selective marker sequence; and the like, as necessary.
[0064] Further, by adding, as necessary, a signal peptide sequence to be localized in a
nucleus, a mitochondria, or an endoplasmic reticulum or immediately below a plasma
membrane, it is possible to localize the fusion protein of the present invention and
accordingly control an intracellular location where the fusion protein functions as
an intrabody.
[0065] The polynucleotide of the present invention may be integrated into a vector. The
term "vector" refers to a vehicle for causing a desired polynucleotide to be introduced
into a host cell and expressed in the host cell. Examples of the vector include: a
viral vector; and a nonviral vector such as a plasmid vector, a bacteria vector, a
phage vector, a phagemid vector, and a cosmid vector. Examples of the viral vector
include adenovirus, adeno-associated virus (AAV), retrovirus (RSV, MMTV, MOMLV, and
the like), lentivirus, Sendai virus, herpes simplex virus, and the like. For introduction
of the polynucleotide of the present invention into a cell
in vivo, it is preferable to use a viral vector. For introduction of the polynucleotide of
the present invention into a cell
in vitro, either one of a viral vector and a nonviral vector can be used.
[0066] The vector may contain, in addition to DNA to be expressed, for example, a regulatory
sequence such as a promoter (SV40 promoter, MMTV-LTR promoter, EF1α promoter, CMV
promoter, and the like), an enhancer, a ribosome binding site, a splice signal, a
terminator, and the like; and, as necessary, a selective marker sequence (ampicillin-resistant
gene, kanamycin-resistant gene, streptomycin-resistant gene, chloramphenicol-resistant
gene, and the like) and the like. Each promoter may be a constitutive promoter or
an inducible promoter.
[0067] Construction of an expression vector can be carried out by, for example, a well-known
genetic engineering technique.
<3. Usage>
[Pharmaceutical use]
(Pharmaceutical composition)
[0068] The present invention also provides a pharmaceutical composition containing the above-described
fusion protein, polynucleotide, or vector.
[0069] The pharmaceutical composition of the present invention may further contain a component
other than the above-described fusion protein, polynucleotide, or vector. The component
other than the above-described fusion protein, polynucleotide, or vector is not limited
to any particular one, and can be, for example, a pharmaceutically acceptable carrier,
lubricant, preservative, stabilizer, wetting agent, emulsifier, osmotic pressure controlling
salt, buffer agent, stabilizer, preservative, excipient, antioxidant, viscosity modifier,
coloring agent, flavoring agent, sweetener, or the like. In a case where the pharmaceutical
composition is provided as an aqueous solution, pure water (sterilized water), physiological
saline, phosphate buffered saline, or the like can be used as a carrier. In a case
where the pharmaceutical composition is provided as an appropriate solution other
than the above, an organic ester (e.g., glycol, glycerol, olive oil, or the like)
that can be introduced into a living organism can be used as a carrier.
[0070] The pharmaceutical composition may be contained in a container, a package, a dispenser,
or the like together with an instruction manual.
(Use in treatment and prevention of disease)
[0071] The above-described pharmaceutical composition may be used, for example, for treatment
and/or prevention of a disease associated with an antigen.
[0072] As an aspect of the pharmaceutical composition of the present invention, the polynucleotide
or the vector of the present invention is introduced into a target cell, and the fusion
protein of the present invention is expressed in the target cell so that a function
of an antigen associated with a disease is controlled (i.e., suppressed or activated,
preferably suppressed) by the antigen-binding peptide. This enables treatment and/or
prevention of the disease.
[0073] As another aspect of the pharmaceutical composition of the present invention, a mechanism
for transporting an antibody from an extracellular environment into a cell is used
to cause the fusion protein of the present invention to enter a target cell and function
as an intrabody. This enables treatment and/or prevention of a disease.
[0074] As another aspect of the pharmaceutical composition of the present invention, in
a mechanism (recycling mechanism) in which an antibody moves from an extracellular
environment into a cell and then is transported into the extracellular environment
again, stability of the fusion protein of the present invention in a target cell is
improved so that the fusion protein functions as an antibody inside and/or outside
the cell. This enables treatment and/or prevention of a disease.
[0075] As another aspect of the pharmaceutical composition of the present invention, a cell
which is a collected cell treated so as to be capable of expressing the fusion protein
of the present invention and in which the fusion protein of the present invention
is expressed is administered to a target. This enables treatment and/or prevention
of a disease. In the above aspect, the collected cell may be a homogeneous syngeneic
cell (an autologous cell) that is collected from an individual to which the cell is
to be administered or may be a heterogeneous syngeneic cell (a heterologous cell)
that is collected from an individual different from the individual to which the cell
is to be administered.
[0076] In any aspect, a localization signal for localization may be added so that the fusion
protein of the present invention is localized in a specific organelle when the fusion
protein is expressed in a cell.
[0077] An aspect of the term "treatment" encompasses reduction or alleviation, delaying
of progress, treatment, and the like of at least one symptom associated with a target
disease. An aspect of the term "prevention" encompasses prevention of development
and the like of at least one symptom associated with a target disease.
[0078] A living organism which is a subject of treatment and/or prevention can be, for example,
a human or a non-human animal, more specifically, a vertebrate such as fish, a bird,
and a mammal. The mammal can be a laboratory animal such as a mouse, a rat, a rabbit,
a guinea pig, and a primate other than a human; a pet animal such as a dog and a cat;
a farm animal such as a pig, a cow, a goat, a sheep, and a horse; or a human.
[0079] Exemplary diseases which are treated or prevented include cancers, tumors, nervous
system diseases (central nervous system diseases, peripheral nervous system diseases),
infectious (viral infection, bacterial infection, and the like) diseases, autoimmune
diseases or allergy diseases, inflammatory diseases, and the like.
[0080] Exemplary cancers or tumors include, but are not limited to, lingual cancer, gingival
cancer, malignant lymphoma, malignant melanoma (melanoma), maxillary cancer, nasal
cancer, nasal cavity cancer, laryngeal cancer, pharyngeal cancer, glioma, myeloma,
glioma, neuroblastoma, papillary carcinoma of thyroid, follicular carcinoma of thyroid,
medullary carcinoma of thyroid, primary pulmonary carcinoma, squamous cell carcinoma,
adenocarcinoma, alveolar carcinoma, large cell undifferentiated carcinoma, small cell
undifferentiated carcinoma, carcinoid, testicular tumor, prostatic cancer, breast
cancer (e.g., papillary adenocarcinoma, comedocarcinoma, mucous tumor, medullary carcinoma,
lobular carcinoma, scirrhous carcinosarcoma, metastatic tumor), mammary Paget disease,
mammary sarcoma, bone tumor, thyroid gland cancer, gastric cancer, liver cancer, acute
myelocytic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia,
acute monocytic leukemia, acute lymphocytic leukemia, acute undifferentiated leukemia,
chronic myeloid leukemia, chronic lymphocytic leukemia, adult T cell leukemia, malignant
lymphoma (e.g., lymphosarcoma, reticulum cell sarcoma, Hodgkin disease or the like),
multiple myeloma, primary macroglobulinemia, infantile leukemia, esophageal carcinoma,
gastric cancer, gastrocolic leiomyosarcoma, gastrointestinal malignant lymphoma, pancreas-gall
bladder cancer, duodenal cancer, large bowel cancer, primary cancer of liver (e.g.,
hepatocellular carcinoma, bile duct cancer or the like), hepatoblastoma, uterine intraepithelial
carcinoma, cervical squamous cell carcinoma, adenocarcinoma of uterus, adenosquamous
carcinoma of uterus, uterine body adenocarcinoma, uterine sarcoma, uterine carcinosarcoma,
invasive hydatidiform mole of uterus, malignant chorioepithelioma of uterus, uterine
malignant melanoma, ovarian cancer, mesodermal mixed tumor, renal carcinoma, renal
pelvic transitional cell carcinoma, ureteral transitional cell carcinoma, papillary
carcinoma of urinary bladder, bladder transitional cell carcinoma, urethral squamous
cell carcinoma, urethral adenocarcinoma, Wilms tumor, rhabdomyosarcoma, fibrosarcoma,
osteosarcoma, chondrosarcoma, synovial sarcoma, myxosarcoma, liposarcoma, Ewing sarcoma,
skin squamous cell carcinoma, skin basal cell carcinoma, skin Bowen disease, skin
Paget disease, skin malignant melanoma, malignant mesothelioma, metastatic adenocarcinoma,
metastatic squamous cell carcinoma, metastatic sarcoma, mesothelioma (e.g., pleural
mesothelioma, peritoneal mesothelioma, pericardial mesothelioma or the like) and the
like.
[0081] Exemplary neurodegenerative diseases include, but are not limited to, Parkinson's
disease, Alzheimer's disease, Huntington's disease, prion disease, frontotemporal
dementia, Amyotrophic Lateral Sclerosis (ALS), Spinalbulbar Muscular Atrophy (SBMA
or Kennedy's Disease), Dentatorubropallidoluysian Atrophy (DRPLA), Spinocerebellar
Ataxia (e.g., SCA-1 through SCA-7), dementia, schizophrenia, depression, manic-depressive
psychosis, neurosis, psychosomatic disorder, cerebral infarction, multiple sclerosis,
progressive supranuclear paralysis, multiple system atrophy, spinocerebellar degeneration,
cerebellar degeneration, abnormality in cerebral metabolism, abnormality in cerebral
circulation, autonomic imbalance, various abnormalities in the endocrine system associated
with the central nervous system, sleep disorder, neurological symptoms (nausea, vomiting,
dry mouth, loss of appetite, dizziness, or the like), dyskinesia, learning disability,
and the like.
[0082] Exemplary infectious diseases include, but are not limited to, diseases caused by
infection with a pathogenic virus such as human immunodeficiency virus (HIV), human
T cell leukemia virus (e.g., HTLV-I), hepatitis virus (e.g., hepatitis A, B, C, D,
or E virus), influenza virus, herpes simplex virus, West Nile fever virus, human papillomavirus,
encephalitis virus, or Ebola virus; diseases caused by infection with pathogenic bacteria
such as chlamydia, mycobacteria, or Legionella; diseases caused by infection with
a pathogenic yeast such as aspergillus or Candida; diseases caused by infection by
a pathogenic protozoan such as malarial parasites or
Trypanosoma parasites; and the like.
[0083] A route and method of administration are not particularly limited, and can be selected
as appropriate in accordance with a target disease. The pharmaceutical composition
may be administered to an affected site directly or indirectly. The route and method
of administration may be administration of cells in which the fusion protein of the
present invention has been expressed. In an example, the route of administration may
be oral, intravenous, intramuscular, subcutaneous, intratumoral, rectal, intraarterial,
intraportal, intraventricular, transmucosal, transdermal, intranasal, intraperitoneal,
intrapulmonary, intrauterine, and the like routes. Topical administration, a method
using a gene gun, and the like may also be employed.
[0084] A dose and a frequency of administration of the pharmaceutical composition may be
selected appropriately according to the degree of a symptom, the age, gender, and
body weight of the patient, the administration form, the specific type of disease,
and the like.
[Use as research tool]
(Research reagent)
[0085] The present invention also provides a research reagent containing the above-described
polynucleotide or vector. The research reagent may further contain a component other
than the above-described polynucleotide or vector. The component other than the above-described
polynucleotide or vector may be one described in the above section "(Pharmaceutical
composition)".
[0086] The research reagent may be contained in a container, a package, a dispenser, or
the like together with an instruction manual.
[Usage as research tool]
[0087] The fusion protein of the present invention can be used for analysis of a function
of an antigen (e.g., a protein, a polypeptide, or the like) in a cell. In an example,
an antigen-binding peptide for the antigen whose function is to be analyzed is intracellularly
expressed as the fusion protein of the present invention, so that the function of
the antigen is controlled (preferably, inhibited) to enable the analysis of the function
of the antigen.
[0088] A target cell is not limited to any particular one. Examples of the target cell include
a human cell and a non-human animal cell, and more specific examples of the target
cell include a vertebrate cell such as a fish cell, an avian cell, and a mammalian
cell. Examples of the mammalian cell include a cell of: a laboratory animal such as
a mouse, a rat, a rabbit, a guinea pig, and a primate other than a human; a pet animal
such as a dog and a cat; a farm animal such as a pig, a cow, a goat, a sheep, and
a horse; or a human. The cell may be a cultured cell or an
in vivo cell (an undissociated cell in a living organism). Preferable examples of the cell
encompass a cultured cell of a human, an
in vivo cell of a human, a cultured cell of a non-human disease-model animal, and an
in vivo cell of a non-human disease-model animal.
[0089] In a case of introducing a viral vector into a target cell, the introduction can
be carried out by a method such as suspension of the viral vector in a cell culture
medium. In a case of introducing a nonviral vector into a target cell, the introduction
can be carried out by a method such as electroporation method, microinjection method,
lipofection method, calcium phosphate method, or DEAE dextran method.
[0090] An indicator of an outcome of inhibition of a function of an antigen can be, for
example, a phenotype change manifested in a cell, a tissue, or an individual. Analysis
of a function of an antigen can be carried out in accordance with these indicators.
Examples of an index of a change in a cell include a phenotype change of a cell, for
example, a quantitative and/or qualitative change of a produced substance, a change
in proliferative activity, a change in cell count, a change in morphology, a change
in characteristics, apoptosis induction, and the like. As a produced substance, it
is possible to use a secretory protein, a surface antigen, an intracellular protein,
mRNA, and the like. As a change in morphology, it is possible to use a change in formation
of a cell process and/or the number of cell processes, a change in oblateness, a change
in extensibility/aspect ratio, a change in cell size, a change in internal structure,
a change in atypia/uniformity and cell density of a cell population, and the like.
These changes in morphology can be confirmed by observation under a microscope. As
a change in characteristics, it is possible to use changes in anchorage dependency,
cytokine-dependent responsiveness, hormone dependency, drug resistance, cell motility,
cell migratory activity, pulsatility, an intracellular substance, and the like. Examples
of cell motility include cell infiltration activity and cell migratory activity. As
a change in the intracellular substance, it is possible to use enzyme activity, amount
of mRNA, amount of an intracellular messenger such as Ca
2+ or cAMP, amount of an intracellular protein, and the like. As a tissue-related indicator,
a functional change according to the tissue to be used can serve as a detection indicator.
As a living organism-related indicator, a change in tissue weight, a change in a blood
system (e.g., a change in blood cell count), a change in amount of protein, a change
in enzyme activity, a change in amount of electrolyte, a change in a circulatory system
(e.g., a change in blood pressure or heart rate), and the like can be used.
[0091] A method for measuring these detection indicators is not particularly limited, and
it is possible to use light absorbance, light emission, color, fluorescence, radioactivity,
fluorescence polarization degree, surface plasmon resonance signal, time-resolved
fluorescence, mass, absorption spectrum, light scattering, fluorescence resonance
energy transfer, and the like. These measurement methods are well-known to a person
skilled in the art, and can be selected as appropriate in accordance with a purpose.
For example, absorption spectrum can be measured by a generally used photometer, plate
reader, or the like, light emission can be measured by a luminometer or the like,
and fluorescence can be measured by a fluorometer or the like. Mass can be measured
by a mass spectrometer. Radioactivity can be measured using a measurement device such
as a gamma counter depending on the type of radiation. Fluorescence polarization degree
can be measured using BEACON (Takara Shuzo Co., Ltd.). Surface plasmon resonance signal
can be measured using BIACORE. Time-resolved fluorescence, fluorescence resonance
energy transfer, and the like can be measured using ARVO or the like. Further, a flow
cytometer and the like can be used for measurement. These methods of measurement can
be used in such a manner that two or more types of detection indicators are measured
by one measurement method, and if convenient, two or more types of measurements can
be performed simultaneously and/or continuously to enable measuring a greater number
of detection indicators. For example, fluorescence and fluorescence resonance energy
transfer can be simultaneously measured using a fluorometer.
[0092] The fusion protein of the present invention can be used for observation of a behavior
of an antigen (e.g., a protein, a polypeptide, or the like) in a cell. In an example,
the observation of the behavior of the antigen can be carried out in such a manner
that (i) an antigen-binding peptide for the antigen whose behavior is to be observed
is intracellularly expressed as the fusion protein of the present invention and (ii)
a reaction between the antigen and the fusion protein is detected. The observation
of the behavior of the antigen encompasses observation of expression of the antigen,
localization of the antigen, and the like at a certain point in time or over time.
[0093] The detection of the reaction between the antigen and the fusion protein can be carried
out, for example, by using fluorescence, light emission, color, or the like of a detection
peptide contained in the fusion protein.
<4. Method for intracellular expression of antigen-binding peptide>
[0094] The present invention also provides a method for intracellular expression of an antigen-binding
peptide. This expression method includes a step of causing the above-described polynucleotide
(i.e., a polynucleotide encoding the fusion protein of the present invention) to be
expressed in a cell.
[0095] The expression may be constitutive expression or transient expression. The expression
may also be inducible expression. For constitutive expression, a polynucleotide in
which a promoter capable of constitutive expression is linked to an upstream gene
of the fusion protein may be used. For transient expression, for example, RNA instead
of DNA may be introduced into and expressed in a cell. For inducible expression, a
polynucleotide in which an inducible promoter is linked to an upstream gene of the
fusion protein may be used. Then, an inducing factor (e.g., IPTG or the like) may
be taken into the cell when the antigen-binding peptide is expressed.
[0096] Examples of a target cell include the cells listed in the section "<3. Usage>".
[0097] In the expression method of the present invention, the antigen-binding peptide is
expressed in a state where the antigen-binding peptide is fused with an intracellular-stabilizer
peptide (that is, as a fusion protein). The fusion protein of the present invention,
due to containing the intracellular-stabilizer peptide, has a low isoelectric point
as compared with a case in which the antigen-binding peptide is present alone. This
allows the fusion protein to undergo less aggregation in a cell as compared with a
case in which the antigen-binding peptide is expressed alone in a cell. Thus, the
fusion protein can stably function as an intrabody.
[0098] In an embodiment, the expression method of the present invention further includes
a step of introducing the above-described polynucleotide into a cell. The above-described
polynucleotide may be introduced in the form of a vector. Examples of a specific introduction
method include the methods described in the section "<3. Usage>". As such, the present
invention further provides a method for producing a cell capable of expressing an
antigen-binding peptide in the cell. This production method further includes a step
of introducing the above-described polynucleotide (i.e., a polynucleotide encoding
the fusion protein of the present invention) or the above-described expression vector
into a cell. The present invention further provides a cell capable of expressing the
fusion protein of the present invention.
<5. Kit>
[0099] The present invention also provides a kit for producing the above-described polynucleotide
encoding a fusion protein containing an intracellular-stabilizer peptide and an antigen-binding
peptide, the kit containing a polynucleotide encoding the intracellular-stabilizer
peptide.
[0100] The kit of the present invention may be a versatile kit that allows easily producing,
for a desired antigen-binding peptide, a polynucleotide encoding a fusion protein.
[0101] The polynucleotide of the present invention encoding an intracellular-stabilizer
peptide may be integrated into the above-described vector. Further, the polynucleotide
may contain a factor (e.g., a promoter, a ribosome binding site, a terminator, and
the like) necessary for protein expression, a selective marker, a restriction enzyme
recognition site, or the like.
[0102] Further, the kit of the present invention may further contain at least one of a buffer,
a restriction enzyme, another necessary reagent, an instrument, an instruction manual,
and the like.
[0103] The following will provide Examples to more specifically describe embodiments of
the present invention. As a matter of course, the present invention is not limited
to Examples provided below, and details of the present invention can be realized in
various manners. Further, the present invention is not limited to the embodiments
described above, and it may be varied in various ways within the scope of the appended
claims. Thus, an embodiment based on a combination of technical means disclosed in
different embodiments is encompassed in the technical scope of the present invention.
Furthermore, all of the publications and patents cited in the present specification
are incorporated herein by reference in their entirety.
[Examples]
[Laboratory animals]
[0104] All experimental procedures were carried out in accordance with the guidelines of
the Animal Experiment Committee of RIKEN. Mice which were used were housed on a 12
h:12 h light/dark cycle, with the dark cycle occurring from 20:00 to 8:00.
[Antibodies]
[0105] Antibodies used in this study are listed in Table S1.
[Isolation of antibody-reactive phages]
[0106] An scFv gene was isolated by the Recombinant Phage Antibody System (RPAS) (GE Healthcare).
DNA sequences of scFv-A36 are deposited in the DNA database of Japan (DDBJ) under
accession number AB472376.
[Construction of expression vectors]
[0107] Expression vectors constructed based on PCR using specific primers are described
below.
[Production of recombinant virus vectors]
[0108] ScFv-GFPA36 and scFv-GFPM4 were produced using AAV1 serotype, and s3Flag-scFv-A36-HA
and s3Flag-scFv-M4-HA were produced using AAV9 serotype. Details of AAV vector plasmids
and production of the AAV vector plasmids are described below.
[Stereotaxic injection of AAV vectors]
[0109] Male wild B6 mice or male DAT-cre (+/-) mice (8 weeks old) were anesthetized with
isoflurane (Escain; Mylan) via an anesthetizer (MK-A110; Muromachi) and placed into
a stereotaxic frame (Stereotaxic Just for Mouse, Muromachi). AAV vectors were injected
into the substantia nigra of the right hemisphere (coordinates relative to bregma
in millimeters, AP; -3.08, LR; -1.25, DV; -4.5).
[Purification of recombinant scFv antibodies]
[0110] Recombinant antibody scFv-GFPA36 was expressed in
E. coli strain BL21 and purified under denatured or non-denatured condition using Ni-NTA
agarose chromatography. Recombinant antibody s3Flag-scFv-A36-HA was purified under
non-denatured condition using anti-Flag (M2) antibody-conjugated affinity beads (Sigma)
from E.
coli strain BL21 co-transformed with pT-Trx vector expressing thioredoxin (Trx) to enhance
soluble expression of foreign proteins (Yasukawa et al., 1995).
[Immunofluorescence staining]
[0111] Immunostaining was performed by a standard technique. Immunofluorescence signals
were visualized with Alexa Fluor 488- or Alexa Fluor 594-labeled secondary antibodies
(Invitrogen). Fluorescence-labeled preparations were imaged under a Fluoview FV1000
confocal microscope (Olympus) or a BZ-9000 fluorescence microscope (Keyence).
[Measurement of antibody affinity by ELISA]
[0112] The antibody binding affinity of GST-Syt I-C2A was measured by ELISA as described
below.
[Behavior test]
[0113] Modified rotarod test was performed with previously reported rotarod test (Shiotsuki
et al., 2010) with a modification. In the present study, rotarod for mice (MK-610A,
Muromachi) was equipped with a large rod (9 cm in diameter) covered by anti-slip tapes
(Nitoflon adhesive tapes, No. 903UL, 0.13 mm in thickness, Nitto denko).
[ScFv proteins aligned with scFv-A36]
[0114] Accession number and amino acid residues of scFv proteins aligned with scFv-A36 are
listed in Table S2.
[Laboratory animals]
[0115] Dat+ /
IRES-cre mice (Backman et al., 2006) were purchased from Jackson laboratory. AAV virus vectors
expressing scFv genes were used together with a DAT-Cre mouse line to selectively
express antibody genes in dopamine neurons of substantia nigra. All mice having virus
vectors used for microdialysis and behavioral tests were each a male littermate from
mated heterozygotes. Balb/c, Balb/c-nu, and wild-type C57B6/J mice purchased from
Charles Liver Japan were used for antibody production or experiments using lentivirus
vectors and AAV vectors, and primary dopamine neuron culture.
[Isolation of antibody-reactive phages]
[0116] In scFv, the heavy- and light-chain variable regions of the antibody are fused by
a glycine linker encoded in a single gene. RPAS (GE Healthcare) allows large repertoires
of scFv to be displayed on the surface of M13 phages. Total RNA was isolated from
mouse (Balb/c) spleen immunized with GST-Syt II-C2A (Fukuda et al., 1994; Fukuda et
al., 1999). The heavy- and light-chain variable regions (VH and VL, respectively)
were amplified in two separate reactions by using degenerate primers (GE Healthcare).
The resultant PCR products were joined by a linker encoding a flexible 15 amino acid
chain of (Gly4-Ser)3. The VH-glycine linker-VL complex (scFv) was subcloned into the
Sfi I and
Not I site of the pCANTAB 5E vector (GE Healthcare). Recombinant phage antibodies were
generated by transformation of E.
coli TG-1 cells with a phagemid vector containing scFv cDNA and infection of M13-KO7 helper
phage. Isolation of antibody-reactive phages was performed by biopanning according
to the manufacturer's instruction manual. Log-phase TG-1 cells were infected with
antibody-reactive phage. Individual antibody-displayed phages from the phage library
were screened by ELISA using recombinant GST-Syt II-C2A bound to the microtiter wells.
The antibody-reactive phages were visualized by horseradish peroxidase (HRP)-conjugated
anti-M13 antibody. Sequencing of the antibody-reactive scFv clone (termed scFv-A36)
was performed by an automated DNA sequencer.
[Construction of expressing vectors]
[0117] Based on the scFv-A36 cDNA sequence, two linker primers were designed for PCR amplification
in which Kozak sequence, T7 peptide, and
BamHI restriction enzyme sites were introduced into the 5' flanking region of A36, and
MunI site, hexa histidine residues, and a
Not I site were introduced into the 3' flanking region of A36 (Table 1).
[Table 1]
Kozak (underline) - T7 peptide (bold) - BamHI (broken line) linker primer (SEQ ID NO: 16): |
|
MunI site (double line) - hexa His (italics) - Not I (underline) linker primer (SEQ ID NO: 17): |
|
[0118] Then, the PCR product was directly ligated into the pGEM-T-easy cloning vector (Promega,
Tokyo, Japan), (the resultant product was named pGEM-scFv-A36; A of Fig. 8). An enhanced
green fluorescent protein (EGFP) fragment was obtained from the pEGFP-C 1 vector (Clontech)
by PCR using the following primers (Table 2).
[Table 2]
BamHI (broken line) linker primer (SEQ ID NO: 18): |
|
Bgl II (double line) linker primer (SEQ ID NO: 19): |
|
[0119] Then, the digested EGFP fragment was ligated into the
BamHI site of pGEM-scFv-A36 to produce a vector pGEM-scFv-GFP A36. To construct pET-scFv-GFP
A36, the
BamHI
-NotI digested fragment of pGEM-scFv-GFPA36 was ligated into the
BamHI and
NotI sites of a modified pET3a (M. Fukuda, unpublished data)
E. coli expression vector (Novagen). For transient expression of scFv-GFPA36 driven by a
cytomegalovirus (CMV) promoter in mammalian cells, the
NotI fragment of pGEM-scFv-GFPA36 was ligated into pIRES vector (Invitrogen) to produce
the vector pIRES-scFv-GFP A36. For the construction of scFv-A36 mutants, a DNA fragment
including CDR1 and CDR3 regions of the heavy chain of A36 was amplified by PCR using
the two following degenerate primers (Table 3). Note that N is A, C, G, or T (equimolar).
[Table 3]
HindIII (underline) linker primer (SEQ ID NO: 20): |
|
Bst EII (double line) linker primer (SEQ ID NO: 21): |
|
[0120] Then, the CDR1 and CDR3 mutant fragments were digested using
HindIII and
BstEII, and were ligated into the
HindIII and
Bst EII site of the parental A36. From these DNA fragments, a mutant scFv-displayed phage
library was generated as described above. Multiple alignment of amino acid sequences
of scFvs was performed by CLUSTALW (version. 2.1: http: / / clustalw.ddbj.nig.ac.jp/index.php?lang=ja)
(Thompson et al.,1994). 3×Flag tag (MDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 1)) and HA
tag (YPYDVPDYA (SEQ ID NO: 14)) fused scFv constructs (s3Flag-scFv-HA) were synthesized
and codon-optimized for the expression in mice. For transient expression in mammalian
cells, the s3Flag-scFv-HA fragments were cloned into the pEF-BOS vector (Mizushima
and Nagata, 1990). The s3Flag-scFv-HA fragments were cloned into the pET3a vector
for expression and purification of s3Flag-scFv-HA proteins in
E. coli BL21 cells.
[Cell culture and transfection]
[0121] Primary dopamine neuron cultures were prepared from the ventral mesencephalon of
embryonic-day 13-14 male and female mouse embryos. Briefly, ventral mesencephalon
was dissociated by treatment with trypsin (0.25% for 20 min at 37°C), followed by
trituration with a fire-polished Pasteur pipette in neurobasal medium supplemented
with 10% FBS containing DNase I. Dissociated cells (6 × 10
4) were plated on poly-L-lysine (1 µg/ml)-coated glass coverslips (Fisherbrand, diameter;
12 mm) in a 24-well plates (Iwaki), and then cultured with 500 µl of neurobasal medium
supplemented with B27 (Invitrogen). The cells at 7 days
in vitro (DIV) in each well were infected with 5 µl of AAV vectors (Titer: 1 × 10
11 pg/ml) to express GFP-tagged scFv proteins, and followed by immunocytochemistry or
dopamine release assay 7 days after infection. 293T and COS-7 cells obtained from
RIKEN Bioresource Center Cell Bank (Tsukuba, Japan) were cultured with DMEM supplemented
with 10% FBS. These cells were transfected with expression vectors by Lipofectamine2000
according to the manufacturer's instruction manual (Invitrogen) and were used for
immunochemical analysis 1 day after transfection.
[Purification of recombinant scFv antibodies]
[0122] E. coli strain BL21 (DE3) (Novagen) was used for expression of scFv-GFPA36. The expression
of scFv-GFPA36 was induced by 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) for
3 h at 30°C. ScFv-GFPA36 protein was solubilized with a buffer (8 M urea, 0.1 M NaH
2PO
4, 10 mM Tris-HCl [pH 8.0]) and purified by Ni-NTA Agarose chromatography (Qiagen)
according to the manufacturer's recommendations. Under denaturing conditions, the
column was washed with 4 ml of a buffer (8 M urea, 0.1 M NaH
2PO
4, 10 mM Tris-HCl [pH 6.3]). ScFv-GFPA36 protein was then eluted from the column with
1 ml of a buffer (8 M urea, 0.1 M NaH
2PO
4, 0.01 M Tris-HCl [pH 4.5]). The eluted scFv-GFPA36 protein was dialyzed with a buffer
(10 mM HEPES-KOH; pH 7.2). To purify scFv-GFPA36 under non-denature condition, the
cells expressing scFv-GFPA36 were re-suspended in a PBS buffer containing protease
inhibitor cocktail and sonicated on ice, and then solubilized for 1h at 4°C by the
addition of Triton X-100 (1%). After centrifugation, the supernatant was subjected
to Ni-NTA (nickel-nitrilotriacetic acid, GE Healthcare) chromatography. Expressed
scFv-GFPA36 was eluted from the column with a buffer (10 mM HEPES-KOH; pH 7.2) containing
5 mM Histidine and followed by dialysis against a buffer (10 mM HEPES-KOH; pH 7.2,
150 mM NaCl).
[0123] To purify s3Flag-scFv-A36-HA under non-denatured condition, E.
coli BL21 (DE3) competent cells (Novagen) were co-transformed with pET3a vector (Novagen)
expressing s3Flag-scFv-A36-HA and pT-Trx vector expressing thioredoxin (Trx) to enhance
soluble expression of foreign proteins (Yasukawa et al., 1995). Log phase transformed
cells were induced by the addition of 1 mM IPTG. The cells were collected by centrifugation
3 h after induction. The cells were re-suspended in a PBS buffer containing protease
inhibitor cocktail. The cell suspension was sonicated on ice and solubilized for 1
h at 4°C by the addition of Triton X-100 (1%). After centrifugation and filtration
with 0.45 µm pore filter, the supernatant was subjected to purification chromatography
using anti-Flag (M2) antibody-conjugated affinity beads (Sigma). Expressed s3Flag-scFv-A36-HA
was purified according to the manufacturer's recommendations. s3Flag-scFv-A36-HA was
eluted from the column with a buffer (50mM Tris-HCl, pH 7.4, 150 mM NaCl) containing
3 × Flag peptide (1 mg/ml) and dialyzed against a buffer (20 mM Hepes-KOH, pH 7.2,
50 mM NaCl).
[Measurement of antibody affinity by ELISA]
[0124] Purified GST-Syt I-C2A (0.25 pmol) in buffer was coated in 96-well plates for 16
h at room temperature. Each wells was blocked with 5% skim milk in a PBS buffer for
2 h at room temperature and was incubated with 0.22 nM to 36 nM purified s3Flag-scFv-A36-HA.
The binding was quantified by incubation with mouse anti-Flag (M2) primary antibody
and HRP-conjugated anti-mouse secondary antibody. Specific binding was calculated
by subtracting the binding of s3Flag-scFv-A36-HA to 0.25 pmol GST-coated wells. Nonlinear
regression of the s3Flag-scFv-A36-HA binding data was performed with the Hill-Langmuir
equation,
where B is the concentration of s3Flag-scFv-A36-HA bound to the GST-Syt I-C2A, B
max is the concentration of the total binding sites, [s3Flag-scFv-A36-HA] is the free
s3Flag-scFv-A36-HA concentration, and K
d is the dissociation constant.
[Immunofluorescence staining]
[0125] Immunocytochemistry and immunohistochemistry were performed by standard technique
as follows. The cells were fixed with 4% PFA for 2 min at room temperature, followed
by permeabilization with 0.3% Triton X-100 in PBS for 2 min at room temperature. The
cells were then immediately washed three times with a blocking solution (1% BSA and
0.1% Triton X-100 in PBS), followed by incubation with the blocking solution for 1
h at room temperature and then with the primary antibodies for 2 h at room temperature.
Immunofluorescence signals were visualized by incubation with Alexa Fluor 488- or
Alexa Fluor 594-labeled secondary antibodies (Invitrogen). The primary antibodies
and the secondary antibodies conjugated with Alexa Fluor dyes are listed in Table
S1. In the experiments quantifying the percentage of the cells with intrabodies-formed
aggregates, at least 100 cells in a dish per each experiment were counted for quantification
of percentage of cells with aggregates. The data were obtained from three independent
experiments. For immunohistochemistry, mice were fixed in 4% paraformaldehyde. Cryosections
of the mice's brains (16 µm in thickness) were permeabilized with 0.3% Triton X-100
in PBS for 2 h at room temperature and incubated with primary antibodies in blocking
solution (1% BSA, 0.1% Triton X-100) for 16 h at 4°C. Immunofluorescence signals were
visualized with Alexa Fluor 488- or Alexa Fluor 594-labeled secondary antibodies (Invitrogen).
Fluorescence-labeled preparations were imaged under a Fluoview FV1000 confocal microscope
(Olympus) or a BZ-9000 fluorescence microscope (Keyence).
[Immunoblot analysis]
[0126] Flag-tagged full-length mouse Syt I-XI were prepared as described previously (Fukuda
et al., 1999). Total homogenate of Flag-tagged Syt I-XI transiently expressed in COS-7
cells was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. Purified
scFv-GFPA36 (1.6 µg/ml) was used as the primary antibody. HRP-labeled anti-T7 monoclonal
antibody (1/5000 dilution, Merck Millipore) was used as the secondary antibody. Immunoreactive
bands were visualized by an enhanced chemiluminescence (ECL) detection system (GE
Healthcare). To ensure that equivalent amounts of Flag-tagged Syts have been loaded,
the blots were reprobed with anti-Flag antibody. To investigate whether purified s3Flag-A36-HA
recognized mouse Syt I/II in the brain, the mouse brain tissues were isolated and
homogenized in a homogenization buffer (20 mM HEPES-KOH, pH 7.2, 150 mM NaCl, 0.5%
TrintonX-100, and complete protease inhibitor cocktail; Roche) and agitated for 1h
at 4°C. Total homogenate of 2.5-pg protein was subjected to western blotting using
purified s3Flag-A36-HA as a primary antibody. Immunoreactive bands were visualized
by ECL using anti-Flag antibody (the secondary antibody) and HRP-labeled anti-mouse
antibody. In the experiments using the mouse brain injected with AAV vectors, the
mouse brain tissues were isolated and homogenized in a buffer (PBS, 0.5% NP40, 1 mM
2ME, 2 mM EDTA, and complete protease inhibitor cocktail) and followed by shearing
with 27-G needle syringe, and agitating for 1 h at 4°C. The total homogenate was subjected
to western blotting using anti-TH, anti-Tubulin, anti-Syt I antibodies as primary
antibodies.
[Immunoprecipitation]
[0127] COS-7 cells were cotransfected with pIRES-scFv-GFPA36 or pIRES-scFv-GFPM4 and pEF-BOS-Flag-Syt
I, or control vector (pEF-BOS). Transfection of plasmid DNA was performed by LipofectAMINE
(Invitrogen) according to the manufacturer's recommendations. Two days after transfection,
the cells were scraped and homogenized in a buffer (10 mM HEPES-KOH [pH 7.2], 100
mM NaCl, 1 mM β-mercaptoethanol). The homogenate was centrifuged at 1,200 × g for
5 min at 4°C, and the supernatant was solubilized with a lysis buffer (10 mM HEPES-KOH
[pH 7.2], 0.1% Triton X-100, 100 mM NaCl, 1 mM β-mercaptoethanol) for 1 h at 4°C.
After centrifugation at 20,400 × g for 15 min at 4°C, the supernatant was transferred
to a new tube, and Flag-Syt I or II was immunoprecipitated by anti-Flag antibody (Sigma)
and protein A sepharose (GE Healthcare). After washing the beads with lysis buffer,
the beads were boiled in 1 × SDS sample buffer (62.5 mM Tris-HCl [pH 6.8], 2% SDS,
2% β-mercaptoethanol, 0.001% bromophenol blue [BPB], 10% glycerol). After centrifugation
at 13,000 × g for 5 min, the supernatant was used for western blot analysis. Coimmunoprecipitated
scFv-GFPA36 was first detected by HRP-labeled anti-T7 antibody. Blots were reprobed
with anti-Syt I antibody (Stressgen) to ensure that Flag-Syt I was precipitated. To
examine the interaction of Syt I and intracellular s3Flag-scFv-A36-HA, 293T cells
were cotransfected with pEF-BOS-Syt I and pEF-BOS-s3Flag-scFv-A36-HA or pEF-BOS-s3Flag-scFv-M4-HA
vector using Lipofectamine2000. Twenty-eight hours after transfection, the cells were
scraped, lysed in a buffer (20 mM HEPES-NaOH [pH 7.4], 150 mM NaCl, 0.5% Triton X-100,
protease inhibitor (EGTA-free, Roche)) for 1 h, and followed by shearing with a 27-gauge
needle syringe. The homogenate was centrifuged at 1,5000 rpm for 10 min at 4°C, and
the supernatant was transferred to a new tube containing CaCl
2 (500 µM) and was subjected to immunoprecipitation by anti-HA agarose (Sigma). After
washing the beads with lysis buffer containing 500 µM CaCl
2, the beads were boiled in 1 × SDS sample buffer for 3 min. After centrifugation at
12,000 × rpm for 5 min, the supernatant was used for western blot analysis. Coimmunoprecipitated
Syt I was detected by the primary anti-Syt I (Stressgen) antibody and the secondary
HRP-labeled anti-mouse-IgG Light chain specific antibody. Blots were also probed with
anti-Flag (M2) primary antibody (Sigma) and anti- HRP-labeled anti-mouse-IgG Light
chain specific secondary antibody to ensure that scFv proteins were precipitated.
[Produce of AAV vectors]
[0128] The AAV9/3 vector plasmids contained the human synapsin I promoter (hSynIp), followed
by the inverted cDNA of interest between the double loxP sequence (loxP-lox2722) derived
from pAAV-DIO-eNpHR-YFP vector (addgene: #26966), a 241-bp fragment (FrgH) containing
nucleotides 2519-2760 derived from rat tau 3'-UTR (Brun et al., 2003), a woodchuck
hepatitis virus posttranscriptional regulatory element (WPRE), and a simian virus
40 polyadenylation signal sequence (SV40pA) between the inverted terminal repeats
of the AAV3 genome. The AAV9 vp cDNA with a mutation (T466F) (Gao et al., 2004) was
synthesized as described previously (Petrs-Silva et al., 2011). The recombinant AAV9/3
vectors were produced by transient transfection of HEK293 cells using the vector plasmid,
an AAV3 rep and AAV9 vp expression plasmid, and the adenoviral helper plasmid pHelper
(Agilent Technologies) as described previously (Li et al., 2006), resulting in the
vectors, AAV9/3-hSynIp-DIO-s3Flag-scFv-A36 or -M4-HA. The recombinant viruses were
purified by isolation from two sequential continuous CsCl gradients, and the viral
titers were determined by qRT-PCR.
[0129] The AAV1 vector plasmids contained the hSynIp, followed by the cDNA of interest,
FrgH, WPRE, and SV40pA between the inverted terminal repeats of the AAV1 genome. Recombinant
AAV1 vectors were produced as described above, resulting in the vectors AAV1-hSynIp-scFvGFPA36
or M4.
[Stereotaxic injection of AAV vectors]
[0130] Male wild mice or female DAT-cre (+/-) mice (8 weeks old) were anesthetized with
isoflurane (Escain; Mylan). The skull at injection site was then cut by a drill (Model
1474 Stereotaxic drill, Muromachi). AAV vectors were injected into the substantia
nigra of the right hemisphere (coordinates relative to bregma in millimeters, AP;
-3.08, LR; -1.25, DV; -4.5) using a 33-gauge needle and a 5-µl syringe (Model 75 RN
SYR, Hamilton) controlled by an injection pump (Legato 130, Muromachi) at 0.2 µl min
-1 for 2 µl. The needle was left in place after injection for 10 min. At least 33 days
after injection, the mice were used for microdialysis, behavior test, or immunochemical
analysis.
[Measurement of dopamine release using primary cultured dopamine neurons]
[0131] The primary dopamine neurons 7 days after infection with AAV vectors were washed
two times with 500 µl of a pre-warmed PSS-LK buffer (20 mM HEPES-NaOH, pH 7.4, 140
mM NaCl, 4.7 mM KCl, 2.5 mM CaCl
2, 1.2 mM MgSO
4, 1.2 mM KH
2PO
4, 11 mM glucose), and the cells were then incubated with 200 µl of a PSS-LK buffer
for 5 min at 37°C. After the PSS-LK buffer was collected, the cells were stimulated
with 200 µl of a PSS-HK buffer (20 mM HEPES-NaOH, pH 7.4, 85 mM NaCl, 60 mM KCl, 2.5
mM CaCl
2, 1.2 mM MgSO
4, 1.2 mM KH
2PO
4, 11 mM glucose) for 5 min at 37°C. Collected supernatants were immediately mixed
with 10 µl of 0.1 M perchloric acid (PCA) containing 50 µM EDTA-2Na, and then sonicated
for 30 sec and followed by centrifugation at 20,000 g and 0°C for 15 min. The supernatant
(190 µl) was transferred to a new tube and mixed with 1.7 µl of 5 M CH
3COONa to adjust the pH to approximately 3.0. After the PSS-HK buffer was collected,
the cells were lysed with 200 µl of 5 mM PCA containing 2.5 µM EDTA-2Na. The samples
(200 µl) were sonicated and mixed with 2.4 µl of 5 M CH
3COONa. All samples were then filtered through 0.45 µm polyvinylidene fluoride (PVDF)
micropore filters (Millipore), and the filtrates were analyzed by high-pressure liquid
chromatography (HPLC) coupled to an electrochemical detection system (ECD300, Eicom)
according to a previous report (Kabayama et al., 2013). The amount of dopamine released
from the primary cultured dopamine neurons exposed to highly-concentrated KCl is expressed
as a percentage of the total dopamine contained in the cells.
[In vivo microdialysis in freely moving mice]
[0132] Mice were each anesthetized with isoflurane (Escain; Mylan) via a small-animal anesthetizer
(MK-A110; Muromachi) and placed into a stereotaxic frame (Stereotaxic Just for Mouse,
Muromachi). A hole was made for implantation of a steel guide cannula (AG-4; Eicom)
on the striatum of the right hemisphere (coordinates relative to bregma, AP; +0.6
mm, LR; -2.0 mm, DV; -2.0 mm), and another hole was made to anchor a stabilizing screw.
The guide cannula and the stabilizing screw were fixed with dental cement (Unifast3;
GC). After the dental cement completely dried, a microdialysis probe (A-I-4-02; Eicom)
was inserted into the striatum through the guide. The implantation of microdialysis
probes was completed in 3.5 h to 4.5 h after the mice started to be anesthetized.
Each mouse inserted with a microdialysis probe was placed onto a paper towel (Kimtowel,
Nippon Paper Crecia) in a transparent microdialysis cage (20 cm in length × 20 cm
in width × 21 cm in height) with water and food. On the next day following implantation
surgery, the mouse was placed onto a new paper towel (Comfort200, Nippon Paper Crecia)
in a transparent microdialysis cage without food and water. To measure basal dopamine
release, collection of dialysate was conducted between 10:00 and 11:00 on the next
day following the probe-implantation surgery. The microdialysis probe was continually
perfused with a Ringer's solution containing 147 mM NaCl, 4.02 mM KCl, 2.25 mM CaCl
2 using a syringe pump (ESP-32, Eicom) at a rate of 1 µl min
-1. The dialysate was collected at 4°C automatically at 10 min intervals into a plastic
microtube preloaded with 5 µl of 0.1 M CH
3COOH/250 µM EDTA-2Na using a refrigerated fraction collector (Refrigerated collector
820, Univentor). The collection of dialysate was conducted 16.5 h to 17.5 h after
the implantation of the microdialysis probes. All dialysate samples were stored at
4°C and analyzed by HPLC on the following day.
[Biopsy of brain section]
[0133] The brains were collected immediately after decapitation, and 50-pm-thick frozen
coronal sections were prepared. Circular tissue punches were collected from 12 substantia
nigra sections and 8 striatum sections (4 anterior sections from coordinates, AP;
+0.6 mm, and 4 posterior sections from coordinates, AP; +0.6 mm) using disposable
biopsy needles (Biopsy Punch; Kai Medical) with a diameter of 1.5 mm. The samples
were homogenized in 0.1 M perchloric acid containing 0.1 mM EDTA and centrifuged for
15 min at 20,000 × g at 4°C. The supernatant was then filtered through a 0.22 µm polyvinylidene
fluoride (PVDF) filter (GV Durapore, Millipore), and the filtrate samples were analyzed
by HPLC.
[HPLC]
[0134] The samples derived from primary cultured neurons and biopsy of brain sections were
analyzed by HPLC (ECD300, Eicom) according to a previously reported protocol (Kabayama
et al., 2013). The microdialysis samples were analyzed by HPLC (ECD500, Eicom) according
to the manufacturer's notes.
[Behavior test]
[0135] Rotarod test with a rod (3 cm in diameter) has been generally used for motor skill
learning of mice. A previous study demonstrated in a modified rotarod test for mice
that a steeper learning curve was obtained by the rotarod test employing a combination
of a large drum and slow rotation (9 cm in diameter and 10 rpm) (Shiotsuki et al.,
2010). In the present study, rotarod for mice (MK-610A, Muromachi) was equipped with
a large rod (9 cm in diameter) covered by anti-slip tapes (Nitoflon adhesive tapes,
No. 903UL, 0.13 mm in thickness, Nitto denko). Prior to training sessions, habituation
was performed by keeping each mouse on a stationary drum for 3 min on the first day.
The habituation was repeated every day for 1 min just before the session. The rotation
was set at a slow speed (5 rpm, 2.8 m/min on the surface). The mouse was placed back
on the drum immediately after falling, up to 5 times in one session. The test was
repeated one session a day for five consecutive days. The latency to falling was recorded
manually and the total latencies on the rod before (Day 1) and after (Day 5) training
were analyzed by two-way repeated measures ANOVA.
[Protein sequence analysis]
[0136] Isometric point and net charge at intracellular pH (7.4, 7.03, 6.60) were calculated
from the amino-acid sequence of each intrabody including peptide tags using Protein
calculator v3.4 developed by Chris Putnam ant the Scrips Research Institute (http://protcalc.sourceforge.net/).
[Statistical analysis]
[0137] All the data are representative of at least three independent experiments. The results
are expressed as the mean ± SEM. The data were analyzed by two-tailed unpaired t-test,
two-tailed non-repeated ANOVA followed by Newman-Keuls post hoc multiple comparison
test, or two-way repeated measures ANOVA followed by bonferroni post hoc multiple
comparison test with the GraphPad Prism 4.0 program (GraphPad Software). P < 0.05
was considered statistically significant.
<Example 1: Isolation of antibody specific binding to the C2A domain of Syt I/II>
[0138] First, an ScFv gene encoding an antibody specific for the C2A domain of Syt I/II
(Syt I/II-C2A) was isolated by using a phage display system. To investigate specificity
of isolated scFv-displayed phages, the C2B domain of Syt I/II (Syt I/II-C2B) was used
as a negative control. Enzyme-linked immunosorbent assay (ELISA) demonstrated that
one of the isolated ScFv-displayed phages (termed scFv-A36) bound to glutathione S-transferase
(GST)-Syt II-C2A and did not bind to GST-Syt II-C2B or GST (A of Fig. 1). From cDNA
sequence, an open reading frame (361 amino acids; accession no. AB472376) which contained
723 nucleotides and in which a heavy chain and a light chain were fused with each
other by a glycine linker was revealed (B of Fig. 1, upper panel). To visualize and
to purify expressed scFv-A36, T7, EGFP, and His tags were fused to the amino terminal
or the carboxyl terminal of A36 (termed scFv-GFPA36; B of Fig. 1, lower panel). ScFv-GFPA36
was expressed in E.
coli and was purified through the Ni-NTA column. Western blot analysis showed that the
purified scFv-GFPA36 protein recognized only full-length Syt I/II and did not recognize
the other 9 isoforms exogenously expressed in COS-7 cells (C of Fig. 1). These results
indicated that scFv-GFPA36 is an antibody specific for the Syt I/II-C2A domain. Amino
acid sequences of three CDRs of the light-chain variable region of scFv-A36 were identical
to those of scFv encoding CRA5, which is an antibody for the immunoglobulin E (IgE)
receptor (accession no. BAB82458, data not shown). This suggested that CDRs of the
light chain in scFv-A36 may not play a key role in the determination of binding specificity
with Syt I/II. On the other hand, it is known that heavy chain CDR3 is the most hypervariable
region in antibodies and has the potential to generate approximately 10
14 varieties in humans (Sanz, 1991), thus contributing to the interaction of antigen
(Chothia and Lesk, 1987). In fact, high diversity was found between the amino acid
sequence of heavy chain CDR3 in scFv-A36 and the amino acid sequences of heavy chain
CDR3 in other 4 scFvs deposited in the National Center for Biotechnology Information
(NCBI) DNA database (Fig. 2). This suggested that heavy chain CDR3 of scFv-A36 might
be important for binding specificity. Then, an attempt was made to construct a negative
control antibody from scFv-GFPA36 by PCR-based mutagenesis. Totally 16 amino acids
in heavy chain CDR1 (8 amino acids) and CDR3 (8 amino acids) of A36 were mutated with
degenerate primers. In the ELISA, it was found that one of the isolated mutant ScFv
phage clones (termed scFv-M4) did not bind to the GST-Syt II-C2A protein (D of Fig.
1), and nucleotide sequences in CDR1 and CDR3 of scFv-M4 were determined (Fig. 2).
It was thus determined that the M4 gene was to be used as a negative control (termed
scFv-GFPM4) for further functional analysis of scFv-GFPA36. Next, whether scFv-GFPA36
can be expressed in mammalian cells and bind to intracellular antibody was examined.
ScFv-GFPA36 was coimmunoprecipitated with anti-Flag antibody only when both scFv-GFPA36
and Flag-Syt I were coexpressed in COS-7 cells, whereas scFv-M4 was not coimmunoprecipitated
with anti-Flag antibody (E of Fig. 1). This indicated that the intracellularly expressed
scFv-GFPA36 can bind to expressed Syt I.
<Example 2: Characterization of intracellular scFv-A36 in vitro and in vivo>
[0139] Because Syt I/II act as major Ca
2+ sensor for fast neurotransmitter release in the brain, the inhibitory effect of scFv-A36
on dopamine release was examined next. To express scFv-GFPA36 and scFv-GFPM4, AAV
vectors were constructed (termed AAV-scFv-GFPA36 and AAV-scFv-GFPM4, respectively).
Primary cultured dopamine neurons at DIV7 prepared from substantia nigra of mouse
embryo were infected with the AAV vectors. The cells were fixed and analyzed 7 days
after infection with AAV. Both scFv-GFPA36 and scFv-GFPM4 were diffusely expressed
without aggregates in the neurons (Fig. 3, A through C; scFv-GFPA36, D through F;
scFv-GFPM4). ScFv expression was also confirmed by western blot analysis (G of Fig.
3, top; scFv, middle; Syt I, bottom; Actin). Then, comparison of depolarization-induced
dopamine release was made. In primary cultured cells, depolarization-induced dopamine
release was extracellular Ca
2+ dependent (H of Fig. 3, right column). This Ca
2+-dependency was consistent with previous reports (Rouge-Pont et al., 1999). Depolarization-induced
dopamine release in scFv-GFPA36-expressing cells was significantly lower than in scFv-GFPM4-expressing
cells or non-infected control cells (H of Fig. 3). These results indicated that the
intrabodies can be expressed in primary cultured neurons and scFv-GFPA36 inhibits
the function of the C2A domain of Syt I/II. Next, whether these intrabodies are stably
expressed in neurons of mouse brain was examined. Adult mice (P56) were unilaterally
injected with AAV-scFv-GFPA36 (1.6×10
8 vg/mouse) into the substantia nigra. Four weeks after injection, scFv-GFPA36 expression
was observed in almost all dopamine neurons. However, intense aggregates were observed
in some neurons in substantia nigra compacta (SNc) (A through C of Fig. 4, arrows).
These fluorescence signals were not observed in non-AAV injected SNc of left hemispheres
(D through F of Fig. 4). These results indicated that intrabodies expressing stably
in
in vitro cultured cells do not always express stably
in vivo.
<Example 3: Improvement of stability of intrabodies by tagging of highly negative
charged 3xFlag peptide and modestly negative charged HA peptide>
[0140] There is a positive correlation between the net negative charge of intrabodies at
cytoplasmic pH 7.4 and stability of intrabodies at cytoplasm in mammalian cells (Kvam
et al., 2010). For example, V
HH-H7 intrabody that has a high net negative charge (-5.0) at pH 7.4 is well expressed
at cytoplasm with less aggregates in mammalian cells (Kvam et al., 2010). This suggests
that the stable intracellular expression of intrabodies can be predicted in a case
where the intrabodies have a net negative charge value of less than -5.0 at pH 7.4.
In fact, scFv-GFPA36 and scFv-GFPM4 that have the same net negative charge -5.0 at
pH 7.4 were stably expressed in primary cultured dopamine neurons for 7 days (Fig.
3). However, scFv-GFPA36 and scFv-GFPM4 formed intracellular aggregates in dopamine
neurons of mouse brain (Fig. 4). Under the circumstances, in silico analysis was conducted
in order to design stable intrabodies
in vivo. It has been reported that the resting intracellular pH in hippocampal neurons is
∼7.03-7.35 (Ruffin et al., 2014), and a previous study using pH-sensitive indicator
pHluorin targeted to endosome has revealed that the local cytoplasmic surface pH of
endosome is 6.73±0.08 (Mitsui et al., 2011). In view of this, a comparison was made
between (i) the pI and net charge of scFv-A36 and scFv-M4 and (ii) those of other
peptide tags, at cytoplasmic pH 6.60, 7.03, and 7.40, respectively (A of Fig. 5).
Fusion of T7-, EGFP-, and His-tags to scFv-A36 or scFv-M4 increased the net negative
charge at pH 7.4. However, the net negative charge of scFv-GFPA36 and scFvGFPM4 dramatically
decreased at a lower pH 7.03 and pH 6.6. This suggested the reduced stability of scFv-GFPA36
and sFv-GFPM4 at cytoplasmic pH 7.03 and 6.6. To improve the net charge of intrabodies
even at the lower pH, short peptide tags, 3×Flag tag (MDYKDHDGDYKDHDIDYKDDDDK (SEQ
ID NO: 1); termed s3Flag) and HA tag were tested. These tags themselves have a strong
net negative charge (s3Flag; -7.0) and a modest net negative charge (HA; -2.2), respectively.
From in silico analysis, it was demonstrated that fusion of s3Flag tag and HA tag
to scFv-A36/M4 proteins (termed s3Flag-scFv-HA) drastically increased their net negative
charge even at pH 6.6 (A of Fig. 5). Next, the generality of the effects of s3Flag-
and HA-peptide tagging on the increase in the net negative charge of intrabodies at
the lower pH was investigated using other 94 scFv protein sequences (Table S2) obtained
by NCBI blast search with scFv-A36 protein sequence (Fig. 6). A comparison was made
between the net charges of 94 scFv proteins fused with different peptide tags at pH
7.4 (A of Fig. 6, upper panels) and pH 6.6 (A of Fig. 6, lower panels). The average
of the net negative charges of scFv proteins fused with T7-, EGFP-, and His-tags was
statistically increased compared with that of scFv proteins without peptide tag at
pH 7.4 (upper panels), but not at pH 6.6 (lower panels). In contrast, the average
of the net negative charge of scFv proteins fused with s3Flag- and HA-tags was statistically
increased compared with that of scFv proteins with or without other peptide tags,
both at pH 7.4 and 6.6. Proteins have a net positive charge under a pH below the pI
of the proteins, or have a net negative charge under a pH above the pI of the proteins.
The average of estimated pI of scFv proteins fused with s3Flag tag and HA tag was
significantly lower than those of scFv proteins fused with or without T7 tag, EGFP
tag, and His tag (A of Fig. 5, B of Fig. 6). These indicated that the effects of s3Flag
tag and HA tag on the increase in the net negative charge of scFv proteins at pH 6.6
were caused by the decrease in the pI of the scFv proteins. Further, a correlation
between amino acid identity of scFv proteins to scFv-A36 and the net charge of scFv
proteins fused with s3Flag tag and HA tag at pH 7.4 was tested (C of Fig. 6). No statistical
correlation was found between the amino acid identity and the net charge. This indicates
that structural identity of scFv proteins to scFv-A36 itself does not affect the effect
of the fusion of s3Flag tag and HA tag to other scFv proteins on the increase in the
net negative charge. Taken together, these results indicated that the fusion of s3Flag-
and HA-peptide tags to scFv proteins generally increases the net negative charge of
the scFv proteins at cytoplasmic pH 6.6 to 7.4 by the strong net negative charge of
these peptide tags and by the decrease in the pI of the scFv proteins.
[0141] Next, the stability of scFv-A36 and scFv-M4 intrabodies fused with these peptide
tags at cytoplasm in mammalian cells was examined. Immunocytochemical analysis revealed
that s3Flag-scFv-A36-HA and s3Flag-scFv-M4-HA were diffusely expressed with little
aggregate in HeLa cells (B of Fig. 5, lower panels), whereas intracellular scFv-GFPA36
and scFv-GFPM4 formed aggregates in HeLa cells (B of Fig. 5, upper panels). The average
of s3Flag-scFv-A36-HA- or s3Flag-scFv-M4-HA-expressing cells with aggregates was significantly
decreased compared with that of scFv-GFPA36- or scFv-GFPM4-expressing cells with aggregates
(C of Fig. 5). These results indicated that s3Flag tag and HA tag could improve stability
of intrabodies in the cultured cells.
<Example 4: s3Flag-scFv-A36-HA interacts with Syt I-C2A domain and inhibits Ca2+-dependent interaction between Syt I and Syntaxin1-SNARE domain>
[0142] Subsequently, the biochemical property of s3Flag-scFvA-36-HA construct was examined.
The binding affinity of purified s3Flag-scFv-A36-HA with GST-Syt I-C2A was measured.
S3Flag-scFv-A36-HA was purified from E.
coli using anti-Flag antibody-conjugated affinity beads. ELISA experiments demonstrated
that purified s3Flag-scFv-A36-HA interacted with Syt I-C2A, but not Syt I-C2B (A of
Fig. 7). This specificity of s3Flag-scFv-A36-HA was consistent with the results of
scFv-GFPA36 as shown in Fig. 1. ELISA experiments using different concentration of
s3Flag-scFv-A36-HA demonstrated that kd value of s3Flag-scFv-A36-HA against SytI-C2A
is approximately 11.97 nM (B of Fig. 7). In addition, western blot analysis revealed
that s3Flag-scFv-A36-HA specifically recognized mouse brain Syt I/II (C of Fig. 7).
These results indicated that s3Flag-scFv-A36-HA is a highly specific intrabody having
high affinity. Next, whether intracellular s3Flag-scFv-A36-HA can bind to Syt I in
cells was investigated using 293T cells. Syt I was co-immunoprecipitated with s3Flag-scFv-A36-HA,
not s3Flag-scFv-M4-HA (D of Fig. 7). This indicates that intracellular s3Flag-scFv-A36-HA
can bind to Syt I in mammalian cells. These raise the possibility that s3Flag-scFv-A36-HA
inhibits its interaction. To test this, the cell lysate show in D of Fig. 7 was subjected
to GST-pull down assay. Precipitation was carried out using purified GST-syntaxin1-SNARE
in the presence of 500 µM Ca
2+, and the precipitates were analyzed by western blotting. Co-precipitates of Syt I
with GST-syntaxin1-SNARE in the cells expressing s3Flag-scFv-A36-HA were decreased
by approximately 46.5% compared with the cells expressing s3Flag-scFv-M4-HA (E, F
of Fig. 7). This indicates that intracellular s3Flag-scFv-A36-HA interferes with the
interaction between Syt I and Syntaxin I in the cells by interacting with the C2A
domain of Syt I.
<Example 5: Stable intracellular expression of s3Flag-scFv-HA in vivo>
[0143] Next, whether s3Flag-scFv-HA is stably expressed in neurons of mouse brain was investigated.
AAV9/3-hSynIp-DIO-s3Flag-scFv-A36 (or M4)-HA was injected to the substantia nigra
of right hemisphere in DAT-cre mice. Immunohistochemistry revealed that s3Flag-scFv-A36-HA
and s3Flag-scFv-M4-HA were stably expressed at cytoplasm of dopamine neurons in SNc
and VTA of right hemispheres 33 days after AAV9/3 vectors injection (A through L of
Fig. 8, arrows). These expression patterns were observed in almost all broad range
of substantia nigra region (∼768 µm) (A through J of Fig. 9). Further, expression
of both s3Flag-scFv-A36-HA and s3Flag-scFv-M4-HA in SN was confirmed using western
blot analysis (K of Fig. 9, upper panel). In striatum where striatal neurons receive
long afferent inputs from mid brain dopamine neurons, expression level of s3Flag-scFv-A36-HA
was higher than that of s3Flag-scFv-M4-HA (K of Fig. 9, upper panel). This suggests
that s3Flag-scFv-A36-HA binds to endogenous Syt I and is transported along with Syt
I from cell body to axonal terminals in striatum. In addition, stable expression of
both s3Flag-scFv-A36-HA and s3Flag-scFv-M4-HA was observed 6 months after AAV9/3 injection
without any neuronal damage of dopamine neurons (Fig. 10). To validate the functional
activity of s3Flag-scFv-A36-HA against Syt I in dopamine neurons of SN
in vivo, basal dopamine release in striatum of awake mice 33 days after AAV9/3 injection was
measured using microdialysis. A comparison of dopamine release in striatum was made
among three groups: s3Flag-scFv-A36-HA-expressing mice, s3Flag-scFv-M4-HA-expressing
mice, and non-AAV-injected mice. It was found that dopamine release in striatum of
s3Flag-scFv-A36-HA-expressing mice was significantly decreased compared with that
of s3Flag-scFv-M4-HA-expressing mice (by 55.3%) or no AAV9/3-injected mice (by 52.7%)
(B, C of Fig. 11). However, total amount of dopamine in striatum and substantia nigra
was not statistically different between all three groups (D of Fig. 11). Taken together,
these results indicated that s3Flag-scFv-A36-HA inhibited dopamine release which is
dependent on neural activity and extracellular Ca
2+ from axonal terminal of dopamine neurons.
<Example 6: Inhibition of motor learning by intracellular expression of s3Flag-scFv-A36-HA
in dopamine neurons>
[0144] Parkinson's disease (PD) is a neurodegenerative disorder that is accompanied by motor
deficits including tremor, rigidity, slowness of movement, and postural instability.
Most characteristic features in patients are loss of dopamine neurons mainly in SNc
and a decrease in dopamine concentration in the striatum. Thus, to examine the direct
role of striatum dopamine release in motor behavior, open field test and modified
rotarod test were performed. AAV9/3-hSynIp-DIO-s3Flag-scFv-A36 (or M4)-HA was bilateral
injected into substantia nigra of DAT-cre mice. Four weeks after AAV injection, the
mice were subjected to open field test, and 5 or 6 days later, to modified rotarod
test. There was no difference of total distance, speed of movement, center of time,
and total rearing number between s3Flag-scFv-A36-HA- and s3Flag-scFv-M4-HA-expressing
mice (A through D of Fig. 12). Next, motor learning was examined using rotarod test
as reported previously (Shiotsuki et al., 2010) with a slightly modification (see
the section of experimental procedures). Five days after training, s3Flag-scFv-M4-HA-expressing
mice stayed on the rod for a longer time than s3Flag-scFv-A36-HA-expressing mice (E
of Fig. 12, P = 0.0132). After motor behavior test, the mouse brain was analyzed by
immunohistochemistry. Bilateral expression of 3Flag-scFv-M4-HA and s3Flag-scFv-A36-HA
proteins in DA neurons of substantia nigra was confirmed (F of Fig. 12, arrows).
<Example 7: Evaluation of antitumor activity>
[0145] Next, anti-Kras monoclonal antibody (Y13-259; SEQ ID NO: 22) was fused with s3Flag
and HA to produce s3Flag-scFv-Y13-259-HA (STAND-Y13-259; SEQ ID NO: 23) in a similar
manner to the above Examples, and evaluation was made as to whether or not s3Flag-scFv-Y
13-259-HA had antitumor activity. Since DNA sequence of Y13-259 was not deposited
in the database, a mouse codon-optimized sequence was designed. DNA sequence of STAND-Y13-259
is represented by SEQ ID NO: 24. DNA sequence of myc-Y13-259 is represented by SEQ
ID NO: 25. DNA synthesis was outsourced to Genescript.
(1) In silico analysis
[0146] In designing STAND-Y13-259, in silico analysis was conducted, which demonstrated
that fusion of s3Flag tag and HA tag drastically increased the net negative charge
of scFv-Y13-259 even at pH 6.6 (A of Fig. 13).
(2) Evaluation of binding capacity of STAND-Y13-259
[0147] Next, the binding affinity between STAND-Y13-259 and GST-Kras was measured by ELISA
in a manner described in the above Examples. The measurement confirmed that STAND-Y
13-259 also maintained a high Kras-binding activity (B of Fig. 13). Subsequently,
MIA PaCa2 cells were infected with a vector obtained by integrating only STAND-Y13-259,
myc-Y13-259 (SEQ ID: NO. 26), or scFv-Y13-259 into lentivirus, and were subjected
to immunocytological staining. As a result, it was confirmed that conventional myc-Y13-259
was unstable and did not express or expressed with aggregates in the cells, whereas
STAND-Y13-259 expressed stably (C of Fig. 13). Further, to confirm interaction in
endogenous Kras cells, coimmunoprecipitation using anti-HA antibody was performed,
which confirmed binding between endogenous Kras and STAND-Y 13-259 in MIA PaCa2 cell-derived
samples infected with STAND-Y13-259 (D of Fig. 13).
(3) Evaluation of antitumor activity
[0148] Next, the viability of MIA PaCa2 cells infected using lentivirus cell vectors in
a manner as described above was measured by MTS analysis 4 days after infection. The
measurement indicated that the viability is significantly reduced by the expression
of STAND-Y13-259 (E of Fig. 13). Subsequently, evaluation was made as to whether the
intracellular expression of STAND-Y13-259 could suppress cancer development in a mouse
xenotransplantation model in which pancreas cancer cells had been subcutaneously transplanted.
The above-described STAND-Y13-259-expressing lentivirus vector was administered to
subcutaneous tumor in nude mice once a week for 4 weeks, and it was thus revealed
that the lentivirus vector strongly inhibits cancer (F, G of Fig. 13). Expression
of STAND-Y13-259 in tumor excised 25 days after initial infection was also confirmed
(H of Fig. 13).
<Example 8: Evaluation of other peptide tags>
[0149] To evaluate the performance of other peptide tags, DE2.0 tag and DE5.0 tag were produced,
and the stabilization effect of DE2.0 tag and DE5.0 tag was studied. In a similar
manner to Example 7, the following fusion proteins containing DE2.0 tag (DEQENDYDEPEVNDENQDYDE
(SEQ ID NO: 13)) were produced: DE2.0-Y13-259 (SEQ ID NO: 27) obtained by fusing DE2.0
tag with the N-terminal of an anti-Kras monoclonal antibody-derived peptide (Y13-259;
SEQ ID NO: 22); DE2.0-Y13-259-HA (SEQ ID NO: 28) obtained by further fusing HA peptide
with the C-terminal of DE2.0-Y13-259; DE2.0-Y13-259-PA (SEQ ID NO: 29) obtained by
further fusing PA peptide with the C-terminal of DE2.0-Y13-259; and protein STAND-6E-LYS
obtained by fusing DE2.0 tag with both of the N- and C-terminals of scFV sequence
(scFv-6E) of an antibody (
Barkhordarian et al., 2006: Protein Engineering, Design and Selection, Volume 19,
Issue 11, 1 November 2006, Pages 497-502) specifically binding to aggregated and fibrous human α-synuclein. Heat shock cognate
protein 70 (HSC70)-binding peptide (MARVKKDQAEPLHRKFERQPPG (SEQ ID NO: 31)) as a functional
peptide was further fused with STAND-6E-LYS, at a position downstream of DE2.0 tag
on the C-terminal side, and HA peptide was further added to a position downstream
of HSC70-binding peptide. The full length of the amino acid sequence of STAND-6E-LYS
thus produced is represented by SEQ ID NO: 32.
[0150] As a fusion protein containing DE5.0 tag (DDDEDEDDEDEDEDEDEDEDED (SEQ ID NO: 33)),
DE5.0-6E (amino acid sequence: SEQ ID NO: 35, base sequence: SEQ ID NO: 36) was produced
by fusing DE5.0 tag with the N-terminal of scFv-6E and further fusing T7 tag (SEQ
ID NO: 34: MASMTGGQQMG) with the C-terminal of scFv-6E. All of the DNA encoding these
fusion proteins were human codon-optimized, and were synthesized by GenScript.
[0151] Note that DNA sequences of DE2.0-Y13-259-HA and STAND-6E-LYS are represented by SEQ
ID NO: 30 and SEQ ID NO: 37, respectively.
[Table 4]
DNA and Amino Acid Sequence of STAND-6E-LYS |
DNA sequence: DE2.0 tag (underline), scFv-6E (italics), HSC70-binding peptide (wavy
underline), HA tag (underline in italics), wherein gccaccatgg at 5' terminal is Kozak
sequence and tga at 3' terminal is stop codon: |
|
Amino acid sequence |
|
[Table 5]
DNA and amino acid sequence of DE5.0-6E |
DE5.0-6E |
DNA sequence: DE5.0 tag (underline), scTv-6E (italics), T7 tag (wavy underline) |
|
Amino acid sequence |
|
[0152] From in silico analysis, it was found that a drastic increase in the net negative
charge of scFv-Y13-259 is caused also by fusion of only DE2.0 tag (A of Fig. 13).
It was also found that additional fusion of HA or PA caused further increase in the
net negative charge (A of Fig. 13). From in silico analysis similarly conducted with
respect to scFv-6E, it was demonstrated that the net negative charge of scFv-6E was
drastically increased by fusion with DE2.0 tag, DE2.0 tag, and HA tag or by fusion
with DE5.0 tag (Fig. 15).
[0153] DE2.0-Y13-259-HA-encoding DNA was introduced into MIA PaCa2 cells with use of a lentivirus
vector. The introduction confirmed expression of DE2.0-Y13-259-HA in the cells without
aggregates, and thus confirmed stronger binding of DE2.0-Y13-259-HA to endogenous
Kras in the cells than STAND-Y13-259 (A of Fig. 14). The cancer development inhibitory
effect of the DE2.0-Y13-259-HA-expressing lentivirus vector was evaluated using a
mouse xenotransplantation model in which pancreas cancer cells had been subcutaneously
transplanted, and it was thus revealed that the lentivirus vector strongly inhibited
cancer (B of Fig. 14). These results indicated that the present invention is extremely
effective for the development of a peptide tag capable of causing an antibody to be
stably expressed in cells.
[0154] STAND-6E-LYS-encoding DNA and DE5.0-6E-encoding DNA were cloned into pEF-BOS vector,
which is a mammalian expression vector, to produce expression vectors (STAND-6E-LYS
expression vector; pEF-BOS-STAND-6E-LYS, DE5.0-6E expression vector; pEF-BOS-DE5.0-6E).
STAND-6E-LYS expression vector and DE5.0-6E expression vector were introduced into
HeLa cells with use of Lipofectamin2000, cultured for 18 hours, and then fixed with
4% PFA. STAND-6E-LYS and DE5.0-6E were subjected to staining with HA antibody and
T7 antibody, respectively. It was confirmed that both STAND-6E-LYS and DE5.0-6E were
expressed in the cells without aggregates (Fig. 16).
[0155] Subsequently, the effect of STAND-6E-LYS on α-synuclein aggregation was examined.
α-synuclein aggregation assay was performed with use of α-synuclein Fibril (
Tarutani et al., 2018: Acta Neuropathol Commun. 2018 Apr 18; 6(1): 29.), which was obtained by subjecting human recombinant α-synuclein purified from E.
coli to sonication to make human recombinant α-synuclein fibrous and aggregated. Human-derived
SHSY5Y cells were used in the assay. HSC70-binding peptide integrated into STAND-6E-LYS
consists of HSC70-binding motif (VKKDQ (SEQ ID NO. 38)) and HSC70-binding consensus
motif (KFERQ (SEQ ID NO. 39)) of α-synuclein. It has been demonstrated
in vitro and
in vivo that fusion between a peptide binding to mutant huntingtin protein and a HSC70-binding
peptide allows degradation of mutant huntingtin protein by chaperon-mediated autophagy
(CMA) to be promoted. Provision of a peptide (including scFv) capable of specifically
binding to a target molecule would thus enable CMA-dependent degradation of the target
protein molecule. pEGFP-α-synuclein vector expressing a protein (EGFP-synuclein) obtained
by fusing the full length of human α-synuclein with green fluorescence protein EGFP
was constructed, and was introduced by gene delivery into SHSY5Y cells together with
STAND-6E-LYS expression vector, with use of XtremGENE9 (Sigma). Six hours later, Fibril
protein was introduced into the cells with use of MultiFectam (Promega), cultured
for 3 days, and then fixed with 4% PFA. Then, STAND-6E-LYS was detected with use of
anti-HA antibody, and staining was carried out using anti-phosphorylated α-synuclein
antibody (Mouse, WAKO). The gene delivery and protein introduction by XtremeGENE9
and MultiFectam were performed according to the manufacturers' standard protocols.
The occurrence ratio of the number of cells in which GFP-synuclein was aggregated
into dots and the occurrence ratio of phosphorylated synuclein positive cells were
measured to quantify aggregates caused by Fibril.
[0156] In approximately 14% of cells into which a control vector had been introduced, Fibril
caused EGFP-synuclein to be localized into dots. It was found that these dots of synuclein
were co-stained by anti-PSer129-synuclein antibody which detects phosphorylation of
the 129th amino acid of α-synuclein, which phosphorylation is a marker of the brain
of a patient with Parkinson's disease (Fig. 17). This indicates that aggregated synuclein
which had entered the cells from outside caused aggregation of EGFP-synuclein which
had had a normal structure capable of being expressed in the cells. On the other hand,
aggregation was reduced to 2% in cells expressing STAND-6E-LYS (Fig. 18).
[0157] These results indicated that the present invention was extremely effective for causing
an antigen-binding peptide to be intracellularly expressed without aggregates and
thus maintaining the function of the antigen-binding peptide.
[Table 6]
[0158]
Table S1
Antibody |
Application |
Description |
Source |
Anti-T7 |
WB, ICC |
mouse monoclonal |
Merckmillipere69522 |
HEP-conjugated anti-T7 |
WB |
mouse monoclonal |
Novagen 69048 |
Anti-Flag |
WB, IP, ICC, IHC |
mouse monoclonal |
Sigma M2 |
Anti-Flag-heads |
Purification |
mouse monoclonal |
Sigma F2425 |
Anti-HA |
WB, IP |
mouse monoclonal |
Sigma A2095 |
Anti-HA agarose |
IP |
mouse monoclonal |
Sigma A2095 |
Anti-Myc |
ICC |
rabbit polyclonal |
MBL 562 |
Anti-GST |
IP |
goat polyclonal |
Amersham pharmacia |
HRP-conjugated anti-M13 |
ELISA |
mouse monoclonal |
Amersham pharmacia |
Anti-GFP |
WB, ICC, IHC |
rabbit polyclonal |
MBL 598 |
Anti-Actin |
WB |
rabbit polyclonal |
Sigma A2066 |
Anti-Tubulin |
WB |
mouse polyclonal |
Sigma T9026 |
Anti-MAP2 |
ICC |
mouse monoclonal |
Chemicon MAB3418 |
Anti-SytI |
WB |
mouse monoclonal |
Stressgen SYA148 |
Anti-TH |
WB, ICC, IHC |
chicken polyclonal |
Abcam ab76442 |
[Table 7]
[0159]
Table S2. scFv proteins aligned with scFv-A36: Accession number|: number of amino
acid residues
|ADC53432.1|:3-244 |
|AAC26537.1|:1-228 |
|ADJ00222.1|:3-248 |
|ADB65759-1|:3-242 |
|CCG26105.1|:20-266 |
|ACB88023.1|:1-242 |
|ADC53454.1|:3-243 |
|AAC26550.1|:1-229 |
|AAC26530.1|:1-231 |
|AAC26528.1|:1-228 |
|CAA10318.1|:3-248 |
|ACS12913.1|:3-240 |
|ADB65756.1|:3-244 |
|S41374:1-247 |
|AAL50781.1|:10-234 |
|ADB65755.1|:3-244 |
|AAY8890S.1|:3-240 |
|AAA83267.1|:1-246 |
|ADB65753-1|.3-241 |
|CAA10385.1|:1-243 |
|ACB97617.1|:3-238 |
|AEK20780.1|:3-237 |
|AAC04222.1|:39-276 |
|AAP23214.1|:3-247 |
|AAQ83756.1|:1-248 |
|ACZ65029.1|:3-236 |
|AAY44382-2|:3-245 |
|1DZB|A:1-239 |
|AAC26540.1|:1-226 |
|BAN78505.1|:8-247 |
|ADB65760.1|:3-238 |
|ADN42857.1|:25-265 |
|BAA22843.1|:3-242 |
|CAC14154.1|:2-247 |
|ACN88153.1|:3-241 |
|AAB03768.1|:2-239 |
|AEK20779.1|:3-242 |
|ACN88155.1|:3-241 |
|BAN78507.1|:8-247 |
|1QOK|A:27-268 |
|AAX07566.1|: 3-240 |
|AF074900_1|:3-245 |
|BAT46707.1|:3-240 |
|AAA19165.1|:23-265 |
|AAQ83757.1|:1-241 |
|ADB65757.1|:3-241 |
|AAKS5297.1|AP402255_1:3-243 |
|AAK85298.1|AF402256_1:3-248 |
|AAY88907.1|:3-240 |
|CAD58896.1|:3-233 |
|AAK56283.1|:1-239 |
|ABN79462.1|:2-242 |
|ACA49232.1|:3-247 |
|AAD33867.1|AF141321_1:3-243 |
|ADN42859-1|:25-265 |
|ADC53451.1|:3-248 |
|AAB65160.1|:1-243 |
|AAF82630.1[:3-243 |
|5AAW|A:2-243 |
|AAY44384.2|:3-245 |
|AAD51317.1|:1-241 |
|AAC26549.1|:1-2:32 |
|BAN78506.1|:8-247 |
|ALJ99801.1|:3-251 |
|CAA103S6.1|:1-249 |
|AEX60024.1|:43-282 |
|AAC26545.1|:1-228 |
|ACB97619.1|:3-238 |
|AAC25685-1|:1-239 |
|AAN32896.1|AF488378_1:3-244 |
|ADC5345S.1|:3-238 |
|AAA75173.1|:23-270 |
|3UYP|A:5-246 |
|AAC26546.1|:1-232 |
|AEX57225.1|:58-297 |
|ADB65758.1|:3-241 |
|CAA94521.1|:1-248 |
|AAL25135.1|AF434672_1:1-237 |
|AAC52185.1|:3-242 |
|AAF82631.1|:3-242 |
|AAU10332.1|:22-271 |
|ADC53456.1|:3-246 |
|AAF85943.1|:1-249 |
|AHM25305.1|:3-249 |
|AAU11282.1|:2-240 |
|CCG26106.1|:20-263 |
|CAC42244.1|:3-244 |
|A56446:3-242 |
|ACV91950.1|:2-239 |
|ACN88154.1|:3-241 |
|CAA82617.1|:1-247 |
|AAG44840.1|:1-250 |
|ACN87219.1|:3-243 |
|
|
JC-5322:1-233 |
Industrial Applicability